Functional annotation map of natural compounds in traditional Chinese medicines library: TCMs with myocardial protection as a case.

The chemical complexity of traditional Chinese medicines (TCMs) makes the active and functional annotation of natural compounds challenging. Herein, we developed the TCMs-Compounds Functional Annotation platform (TCMs-CFA) for large-scale predicting active compounds with potential mechanisms from TCM complex system, without isolating and activity testing every single compound one by one. The platform was established based on the integration of TCMs knowledge base, chemome profiling, and high-content imaging. It mainly included: (1) selection of herbal drugs of target based on TCMs knowledge base; (2) chemome profiling of TCMs extract library by LC‒MS; (3) cytological profiling of TCMs extract library by high-content cell-based imaging; (4) active compounds discovery by combining each mass signal and multi-parametric cell phenotypes; (5) construction of functional annotation map for predicting the potential mechanisms of lead compounds. In this stud TCMs with myocardial protection were applied as a case study, and validated for the feasibility and utility of the platform. Seven frequently used herbal drugs (Ginseng, etc.) were screened from 100,000 TCMs formulas for myocardial protection and subsequently prepared as a library of 700 extracts. By using TCMs-CFA platform, 81 lead compounds, including 10 novel bioactive ones, were quickly identified by correlating 8089 mass signals with 170,100 cytological parameters from an extract library. The TCMs-CFA platform described a new evidence-led tool for the rapid discovery process by data mining strategies, which is valuable for novel lead compounds from TCMs. All computations are done through Python and are publicly available on GitHub.

Cardioprotective effects of arjunolic acid in LPS-stimulated H9C2 and C2C12 myotubes via the My88-dependent TLR4 signaling pathway.

CONTEXT: Arjunolic acid (AA) is a triterpenoid saponin found in Terminalia arjuna (Roxb.) Wight & Arn. (Combretaceae). It exerts cardiovascular protective effects as a phytomedicine. However, it is unclear how AA exerts the effects at the molecular level. OBJECTIVE: This study investigates the cardioprotective effects of arjunolic acid (AA) via MyD88-dependant TLR4 downstream signaling marker expression. MATERIALS AND METHODS: The MTT viability assay was used to assess the cytotoxicity of AA. LPS induced in vitro cardiovascular disease model was developed in H9C2 and C2C12 myotubes. The treatment groups were designed such as control (untreated), LPS control, positive control (LPS + pyrrolidine dithiocarbamate (PDTC)-25 µM), and treatment groups were co-treated with LPS and three concentrations of AA (50, 75, and 100 µM) for 24 h. The changes in the expression of TLR4 downstream signaling markers were evaluated through High Content Screening (HCS) and Western Blot (WB) analysis. RESULTS: After 24 h of co-treatment, the expression of TLR4, MyD88, MAPK, JNK, and NF-κB markers were upregulated significantly (2-6 times) in the LPS-treated groups compared to the untreated control in both HCS and WB experiments. Evidently, the HCS analysis revealed that MyD88, NF-κB, p38, and JNK were significantly downregulated in the H9C2 myotube in the AA treated groups. In HCS, the expression of NF-κB was downregulated in C2C12. Additionally, TLR4 expression was downregulated in both H9C2 and C2C12 myotubes in the WB experiment. DISCUSSION AND CONCLUSIONS: TLR4 marker expression in H9C2 and C2C12 myotubes was subsequently decreased by AA treatment, suggesting possible cardioprotective effects of AA.

TORNADO-seq: A Protocol for High-Throughput Targeted RNA-seq-Based Drug Screening in Organoids.

Organoids are 3D ex vivo cell aggregates derived from primary tissue and shown to closely recapitulate tissue homeostasis. Organoids deliver certain advantages compared to 2D cell lines and mouse models, especially in drug-screening studies and translational research projects. The application of organoids in the research field is fast-emerging and new techniques for organoid manipulation are constantly developing. Despite recent advances, RNA-seq-based drug-screening platforms in organoids are not yet established. Here, we provide a detailed protocol for performing TORNADO-seq, a targeted RNA-seq-based drug-screening method in organoids. Analyzing complex phenotypes with a large number of carefully selected read-outs allows to directly classify and group drugs even without structural similarity or overlapping mode of actions from prior knowledge. Our assay principle combines cost-effectiveness and sensitive detection of multiple cell identities, signaling pathways, and key drivers of cellular phenotypes and can be applied to many systems where this new form of high-content screening can provide information not obtainable otherwise.

The latest advances in high content screening in microfluidic devices.

INTRODUCTION: High content screening (HCS) is an important tool for drug screening. However, the potential of HCS in the field of drug screening and synthetic biology is limited by traditional culture platforms that use multi-well plates, which have several disadvantages. Recently, microfluidic devices have gradually been applied in HCS, which significantly reduces experimental costs, increases assay throughput, and improves the accuracy of drug screening. AREAS COVERED: This review provides an overview of microfluidic devices for high-content screening in drug discovery platforms, including droplet, microarray, and organs-on-chip technologies. EXPERT OPINION: HCS is a promising technology increasingly adopted by the pharmaceutical industry as well as academic researchers for drug discovery and screening. In particular, microfluidic-based HCS shows unique advantages, and microfluidics technology has promoted significant advancements and broader usage and applicability of HCS in drug discovery. With the integration of stem cell, gene editing technology, and other biological technologies, microfluidics-based HCS will expand the application scope of personalized disease and drug screening models. The authors anticipate rapid developments in this field, with microfluidic-based approaches becoming increasingly important in HCS applications.

High-content screening of active components of Traditional Chinese Medicine inhibiting TGF-ß-induced cell EMT.

The epithelial mesenchymal transition (EMT) has roles in metastasis and invasion during fibrotic diseases and cancer progression. Some Traditional Chinese Medicines (TCMs) have shown inhibitory effects with respect to the EMT. The current study attempted to establish a multiparametric high-content method to screen for active monomeric compounds in TCM with the ability to target cellular EMT by assessing phenotypic changes. A total of 306 monomeric compounds from the MedChemExpress (MCE) compound library were screened by the high-content screening (HCS) system and 5 compounds with anti-EMT activity, including camptothecin (CPT), dimethyl curcumin (DMC), artesunate (ART), sinapine (SNP) and berberine (BER) were identified. To confirm anti-EMT activity, expression of EMT markers was assessed by qRT-PCR and Western blotting, and cell adhesion and migration measured by cell function assays. The results revealed that CPT, DMC, ART, SNP and BER inhibited transforming growth factor-ß1 (TGF-ß1)-induced expression of vimentin and α-SMA, upregulated expression of E-cadherin, increased cell adhesion and reduced cell migration. In summary, by quantifying the cell morphological changes during TGF-ß1-induced EMT through multi-parametric analysis, TCM compounds with anti-EMT activity were successfully screened using the HCS system, a faster and more economical approach than conventional methods.

The interaction of alkaloids in Coptis chinensis Franch -Tetradium ruticarpum (A. Juss.) T.G. Hartley with hOCT1 and hOCT2.

ETHNOPHARMACOLOGICAL RELEVANCE: Zuojin Pill, a traditional poly-herbal drug, comprises Coptis chinensis Franch - Tetradium ruticarpum (A. Juss.) T.G. Hartley (6:1). The significant quantity of alkaloids found in the participating herbs is a key aspect of the Zuojin Pill. According to traditional Chinese medicine (TCM), these numerous alkaloidal compounds within Zuojin Pill have various essential therapeutic effects. AIM OF THE STUDY: The alkaloids in Tetradium are mainly indole alkaloids, while the alkaloids in Coptis are mostly isoquinoline alkaloids with low bioavailability. Alkaloids and their metabolites are nitrogen-containing compounds or weakly alkaline substances that can be partially ionized under physiological pH conditions. Fortunately, organic cation transporters (OCTs) play a crucial role in the cellular uptake of weakly alkaline compounds. Therefore, we speculated that the alkaloidal compounds might interact with liver cation transporters hOCT1 and kidney cation transporters hOCT2 to alter cell drug disposal. In order to clarify our hypothesis, a series of alkaloids-OCTs interaction experiments were conducted. MATERIALS AND METHODS: HEK293 cells stably expressing hOCT1 and hOCT2 were modeled and evaluated. Afterward, high-content screening (HCS) was conducted to analyze whether the main alkaloids and their metabolites of Coptis - Tetradium were inhibitors of hOCT1 and hOCT2 transporters. Meanwhile, LC-MS/MS was used to investigate whether the alkaloidal compounds were substrates of hOCT1 and hOCT2 transporters. Finally, drug interactions at the cellular level were assessed by LC-MS/MS after co-administration of berberine and rutacorine. RESULTS: Berberine, jateorhizine, coptisine, epiberberine, columbamine, demethyleneberberine, and berberrubine could significantly inhibit hOCT1 and hOCT2 activity. Isoquinoline alkaloids, including berberine, jateorhizine, coptisine, epiberberine, columbamine, and palmatine, were substrates of hOCT1 and hOCT2, but not the indole alkaloids evodiamine and rutaecarpine. Furthermore, evodiamine at a concentration of 20 µmol/L had a trivial effect on berberine accumulation in HEK293-hOCT2 cells. CONCLUSIONS: These results support the idea that alkaloidal compounds within Coptis and Tetradium have hOCT1 and hOCT2 inhibitory activity or be their substrates, and the increased oral bioavailability of berberine in vivo was closely related to the potential interactions of small molecules in Coptis- Tetradium. Overall, our study provides a framework for investigating the potential interactions of small molecules in Coptis- Tetradium.

Human Sensory Neuron-like Cells and Glycated Collagen Matrix as a Model for the Screening of Analgesic Compounds.

Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (ß-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.

Drug Repurposing in Rare Diseases: An Integrative Study of Drug Screening and Transcriptomic Analysis in Nephropathic Cystinosis.

Diagnosis and cure for rare diseases represent a great challenge for the scientific community who often comes up against the complexity and heterogeneity of clinical picture associated to a high cost and time-consuming drug development processes. Here we show a drug repurposing strategy applied to nephropathic cystinosis, a rare inherited disorder belonging to the lysosomal storage diseases. This approach consists in combining mechanism-based and cell-based screenings, coupled with an affordable computational analysis, which could result very useful to predict therapeutic responses at both molecular and system levels. Then, we identified potential drugs and metabolic pathways relevant for the pathophysiology of nephropathic cystinosis by comparing gene-expression signature of drugs that share common mechanisms of action or that involve similar pathways with the disease gene-expression signature achieved with RNA-seq.

Identification of Insulin-Mimetic Plant Extracts: From an In Vitro High-Content Screen to Blood Glucose Reduction in Live Animals.

Type 2 diabetes mellitus (T2DM) is linked to insulin resistance and a loss of insulin sensitivity, leading to millions of deaths worldwide each year. T2DM is caused by reduced uptake of glucose facilitated by glucose transporter 4 (GLUT4) in muscle and adipose tissue due to decreased intracellular translocation of GLUT4-containing vesicles to the plasma membrane. To treat T2DM, novel medications are required. Through a fluorescence microscopy-based high-content screen, we tested more than 600 plant extracts for their potential to induce GLUT4 translocation in the absence of insulin. The primary screen in CHO-K1 cells resulted in 30 positive hits, which were further investigated in HeLa and 3T3-L1 cells. In addition, full plasma membrane insertion was examined by immunostaining of the first extracellular loop of GLUT4. The application of appropriate inhibitors identified PI3 kinase as the most important signal transduction target relevant for GLUT4 translocation. Finally, from the most effective hits in vitro, four extracts effectively reduced blood glucose levels in chicken embryos (in ovo), indicating their applicability as antidiabetic pharmaceuticals or nutraceuticals.

Identification of anti-inflammatory compounds from Zhongjing formulae by knowledge mining and high-content screening in a zebrafish model of inflammatory bowel diseases.

BACKGROUND: Inflammatory bowel diseases (IBD) are chronic relapsing intestinal inflammations with increasing global incidence, and new drug development remains in urgent demand for IBD management. To identify effective traditional Chinese medicine (TCM) formulae and compounds in IBD treatment, we innovatively combined the techniques of knowledge mining, high-content screening and high-resolution mass spectrometry, to conduct a systematic screening in Zhongjing formulae, which is a large collection of TCM prescriptions with most abundant clinical evidences. METHODS: Using Word2vec-based text learning, the correlations between 248 Zhongjing formulae and IBD typical symptoms were analyzed. Next, from the top three formulae with predicted relationship with IBD, TCM fractions were prepared and screened on a transgenic zebrafish IBD model for their therapeutic effects. Subsequently, the chemical compositions of the fraction hits were analyzed by mass spectrometry, and the major compounds were further studied for their anti-IBD effects and potential mechanisms. RESULTS: Through knowledge mining, Peach Blossom Decoction, Pulsatilla Decoction, and Gegen Qinlian Decoction were predicted to be the three Zhongjing formulae mostly related to symptoms typical of IBD. Seventy-four fractions were prepared from the three formulae and screened in TNBS-induced zebrafish IBD model by high-content analysis, with the inhibition on the intestinal neutrophil accumulation and ROS level quantified as the screening criteria. Six herbal fractions showed significant effects on both pathological processes, which were subsequently analyzed by mass spectrometry to determine their chemical composition. Based on the major compounds identified by mass spectrometry, a second-round screen was conducted and six compounds (palmatine, daidzin, oroxyloside, chlorogenic acid, baicalin, aesculin) showed strong inhibitory effects on the intestinal inflammation phenotypes. The expression of multiple inflammatory factors, including il1ß, clcx8a, mmp and tnfα, were increased in TNBS-treated fish, which were variously inhibited by the compounds, with aesculin showing the most potent effects. Moreover, aesculin and daidzin also upregulated e-cadherin's expression. CONCLUSION: Taken together, we demonstrated the regulatory effects of several TCM formulae and their active compounds in the treatment of IBD, through a highly efficient research strategy, which can be applied in the discovery of effective TCM formulae and components in other diseases.

A High-Content Screening Assay for Small Molecules That Stabilize Mutant Triose Phosphate Isomerase (TPI) as Treatments for TPI Deficiency.

Triose phosphate isomerase deficiency (TPI Df) is an untreatable, childhood-onset glycolytic enzymopathy. Patients typically present with frequent infections, anemia, and muscle weakness that quickly progresses with severe neuromusclar dysfunction requiring aided mobility and often respiratory support. Life expectancy after diagnosis is typically ~5 years. There are several described pathogenic mutations that encode functional proteins; however, these proteins, which include the protein resulting from the "common" TPIE105D mutation, are unstable due to active degradation by protein quality control (PQC) pathways. Previous work has shown that elevating mutant TPI levels by genetic or pharmacological intervention can ameliorate symptoms of TPI Df in fruit flies. To identify compounds that increase levels of mutant TPI, we have developed a human embryonic kidney (HEK) stable knock-in model expressing the common TPI Df protein fused with green fluorescent protein (HEK TPIE105D-GFP). To directly address the need for lead TPI Df therapeutics, these cells were developed into an optical drug discovery platform that was implemented for high-throughput screening (HTS) and validated in 3-day variability tests, meeting HTS standards. We initially used this assay to screen the 446-member National Institutes of Health (NIH) Clinical Collection and validated two of the hits in dose-response, by limited structure-activity relationship studies with a small number of analogs, and in an orthogonal, non-optical assay in patient fibroblasts. The data form the basis for a large-scale phenotypic screening effort to discover compounds that stabilize TPI as treatments for this devastating childhood disease.

The Use of Informer Sets in Screening: Perspectives on an Efficient Strategy to Identify New Probes.

Small-molecule discovery typically involves large-scale screening campaigns, spanning multiple compound collections. However, such activities can be cost- or time-prohibitive, especially when using complex assay systems, limiting the number of compounds tested. Further, low hit rates can make the process inefficient. Sparse coverage of chemical structure or biological activity space can lead to limited success in a primary screen and represents a missed opportunity by virtue of selecting the "wrong" compounds to test. Thus, the choice of screening collections becomes of paramount importance. In this perspective, we discuss the utility of generating "informer sets" for small-molecule discovery, and how this strategy can be leveraged to prioritize probe candidates. While many researchers may assume that informer sets are focused on particular targets (e.g., kinases) or processes (e.g., autophagy), efforts to assemble informer sets based on historical bioactivity or successful human exposure (e.g., repurposing collections) have shown promise as well. Another method for generating informer sets is based on chemical structure, particularly when the compounds have unknown activities and targets. We describe our efforts to screen an informer set representing a collection of 100,000 small molecules synthesized through diversity-oriented synthesis (DOS). This process enables researchers to identify activity early and more extensively screen only a few chemical scaffolds, rather than the entire collection. This elegant and economic outcome is a goal of the informer set approach. Here, we aim not only to shed light on this process, but also to promote the use of informer sets more widely in small-molecule discovery projects.

The Scar-in-a-Jar: In Vitro Fibrosis Model for Anti-Fibrotic Drug Testing.

Excessive deposition of type I collagen follows in the wake of chronic inflammation processes in dysregulated tissue healing and causes fibrosis that can ultimately lead to organ failure. While the development of antifibrotic drugs is targeting various upstream events in collagen matrix formation (synthesis, secretion, deposition, stabilization, remodeling), the evaluation of drug effects would use as net read-out of the above effects the presence of a deposited collagen matrix by activated cells, mainly myofibroblasts. Conventional methods comprise lengthy and labor-intensive protocols for the quantification of deposited collagen, some with sensitivity and/or specificity issues. Here we describe the Scar-in-a-Jar assay, an in vitro fibrosis model for anti-fibrotic drug testing that benefits from a substantially accelerated extracellular matrix deposition employing macromolecular crowding and a collagen-producing cell type of choice (e.g., lung fibroblasts like WI-38). The system can be aided by activating compounds such as transforming growth factor-ß1, a classical inducer of the myofibroblast phenotype in fibroblasts. Direct image analysis of the well plate not only eliminates the need for matrix extraction or solubilization methods, but also allows for direct imaging and monitoring of phenotypical markers and offers the option for high-content screening applications when adapted to well formats compatible with a screening format.

Screening of FDA-Approved Drugs Using a MERS-CoV Clinical Isolate from South Korea Identifies Potential Therapeutic Options for COVID-19.

Therapeutic options for coronaviruses remain limited. To address this unmet medical need, we screened 5406 compounds, including United States Food and Drug Administration (FDA)-approved drugs and bioactives, for activity against a South Korean Middle East respiratory syndrome coronavirus (MERS-CoV) clinical isolate. Among 221 identified hits, 54 had therapeutic indexes (TI) greater than 6, representing effective drugs. The time-of-addition studies with selected drugs demonstrated eight and four FDA-approved drugs which acted on the early and late stages of the viral life cycle, respectively. Confirmed hits included several cardiotonic agents (TI > 100), atovaquone, an anti-malarial (TI > 34), and ciclesonide, an inhalable corticosteroid (TI > 6). Furthermore, utilizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we tested combinations of remdesivir with selected drugs in Vero-E6 and Calu-3 cells, in lung organoids, and identified ciclesonide, nelfinavir, and camostat to be at least additive in vitro. Our results identify potential therapeutic options for MERS-CoV infections, and provide a basis to treat coronavirus disease 2019 (COVID-19) and other coronavirus-related illnesses.

Development and validation of a high-content screening assay for inhibitors of enteropathogenic E. coli adhesion.

Enteropathogenic E. coli (EPEC) causes intestinal infections leading to severe diarrhea. EPEC attaches to the host cell causing lesions to the intestinal epithelium coupled with the effacement of microvilli. In the process, actin accumulates into a pedestal-like structure under bacterial microcolonies. We designed an automated fluorescence microscopy-based screening method for discovering compounds capable of inhibiting EPEC adhesion and virulence using aurodox, a type three secretion system (T3SS) inhibitor, as a positive control. The screening assay employs an EPEC strain (2348/69) expressing a fluorescent protein and actin staining for monitoring the bacteria and their pedestals respectively, analyzing these with a custom image analysis pipeline. The assay allows for the discovery of compounds capable of preventing the formation of pathogenic actin rearrangements. These compounds may be interfering with virulence-related molecular pathways relevant for developing antivirulence leads.

A New Method for Screening Natural Products to Stimulate IFN-γ Production in Jurkat Human T Lymphocytes.

Interferon-γ (IFN-γ) is a critical cytokine in the defense against viral and bacterial infection. It is mainly produced by natural killer cells and activated T cells. Given its regulatory role in coordinating cellular and humoral immune responses, IFN-γ is considered to be an effective therapeutic agent in the treatment of viral infection. Here we established a fluorescence-based high-content screening model to find small molecules that can stimulate the production of IFN-γ in human Jurkat cells. After a primary screening of 267 natural products, two hits, Astragalus polyphenols and 6-shogaol, were identified to promote the activity of the IFN-γ promoter and subsequently validated by the flow cytometry assay. Obviously, both Astragalus polyphenols and 6-shogaol exhibited potential to induce the transcription and expression of IFN-γ in a dose-dependent manner. These results indicated that our high-content screening model could be a credible and useful platform to contribute to the discovery of novel molecules to promote the expression of IFN-γ and provide leading compounds for the treatment of viral infectious diseases.

High-Content Drug Discovery Screening of Endocytosis Pathways.

Endocytosis is the dynamic internalization of cargo (receptors, hormones, viruses) for cellular signaling or processing. It involves multiple mechanisms, classified depending on critical proteins involved, speed, morphology of the derived intracellular vesicles, or substance trafficked. Pharmacological targeting of specific endocytosis pathways has a proven utility for diverse clinical applications from epilepsy to cancer. A multiplexable, high-content screening assay has been designed and implemented to assess various forms of endocytic trafficking and the associated impact of potential small molecule modulators. The applications of this assay include (1) drug discovery in the search for specific, cell-permeable endocytosis pathway inhibitors (and associated analogues from structure-activity relationship studies), (2) deciphering the mechanism of internalization for a novel ligand (using pathway-specific inhibitors), (3) assessment of the importance of specific proteins in the trafficking process (using CRISPR-Cas9 technology, siRNA treatment, or transfection), and (4) identifying whether endocytosis inhibition is an off-target for novel compounds designed for alternative purposes. We describe this method in detail and provide a range of troubleshooting options and alternatives to modify the protocol for lab-specific applications.

Androgen Signaling Regulates SARS-CoV-2 Receptor Levels and Is Associated with Severe COVID-19 Symptoms in Men.

SARS-CoV-2 infection has led to a global health crisis, and yet our understanding of the disease and potential treatment options remains limited. The infection occurs through binding of the virus with angiotensin converting enzyme 2 (ACE2) on the cell membrane. Here, we established a screening strategy to identify drugs that reduce ACE2 levels in human embryonic stem cell (hESC)-derived cardiac cells and lung organoids. Target analysis of hit compounds revealed androgen signaling as a key modulator of ACE2 levels. Treatment with antiandrogenic drugs reduced ACE2 expression and protected hESC-derived lung organoids against SARS-CoV-2 infection. Finally, clinical data on COVID-19 patients demonstrated that prostate diseases, which are linked to elevated androgen, are significant risk factors and that genetic variants that increase androgen levels are associated with higher disease severity. These findings offer insights on the mechanism of disproportionate disease susceptibility in men and identify antiandrogenic drugs as candidate therapeutics for COVID-19.

Patient-Derived Induced Pluripotent Stem Cell-Based Models in Parkinson's Disease for Drug Identification.

Parkinson's disease (PD) is a common progressive neurodegenerative disorder characterized by loss of striatal-projecting dopaminergic neurons of the ventral forebrain, resulting in motor and cognitive deficits. Despite extensive efforts in understanding PD pathogenesis, no disease-modifying drugs exist. Recent advances in cell reprogramming technologies have facilitated the generation of patient-derived models for sporadic or familial PD and the identification of early, potentially triggering, pathological phenotypes while they provide amenable systems for drug discovery. Emerging developments highlight the enhanced potential of using more sophisticated cellular systems, including neuronal and glial co-cultures as well as three-dimensional systems that better simulate the human pathophysiology. In combination with high-throughput high-content screening technologies, these approaches open new perspectives for the identification of disease-modifying compounds. In this review, we discuss current advances and the challenges ahead in the use of patient-derived induced pluripotent stem cells for drug discovery in PD. We address new concepts implicating non-neuronal cells in disease pathogenesis and highlight the necessity for functional assays, such as calcium imaging and multi-electrode array recordings, to predict drug efficacy. Finally, we argue that artificial intelligence technologies will be pivotal for analysis of the large and complex data sets obtained, becoming game-changers in the process of drug discovery.

A two-dimensional multiwell cell culture method for the production of CYP3A4-expressing hepatocyte-like cells from HepaRG cells.

Cytochrome P450 enzymes (CYP) function in drug metabolism in the liver. To evaluate numerous drug candidates, a high-content screening (HCS) system with hepatocyte-like cells (HLCs) that can replace adult human hepatocytes is required. Human hepatocellular carcinoma HepaRG is the only cell line capable of providing HLCs with high CYP3A4 expression comparable to that in adult hepatocytes after cell differentiation. The aim of this study was to design an ideal multiwell culture system for HLCs using transgenic HepaRG cells expressing the EGFP coding an enhanced green fluorescent protein under CYP3A4 transcriptional regulation. HLCs were matured on five different types of 96-well black plates. Culturing HLCs on glass-bottom Optical CVG plates significantly promoted cell maturation and increased metabolic activity by twofold under two-dimensional (2D) culture conditions, and these features were enhanced by 2% collagen coating. Three plates for three-dimensional (3D) cell cultures with a gas-exchangeable fabric or dimethylpolysiloxane membrane bottom formed multiple round colonies, whereas they were ineffective for CYP3A4 expression. Under optimized conditions presented here, HLCs lost responsiveness to nuclear receptor-mediated transcriptional induction of CYP3A4, suggesting that CYP3A4 transcription has already been fully upregulated. Therefore, HepaRG-derived HLCs will provide an alternative to human hepatocytes with high levels of CYP3A4 enzyme activity even under 2D culture conditions. This will improve a variety of drug screening methods.