96-well Format Research Articles

Quantitative Biochemical Analysis of Deadenylase Enzymes Using Fluorescence and Chemiluminescence-Based Assays.

Deadenylase enzymes play a key role in mRNA degradation and RNA processing. In this chapter, we describe two activity assays for the quantitative biochemical analysis of deadenylase enzymes, which can easily be adapted for other nuclease enzymes. The assays use distinct principles of detection, which are based on differential annealing of a probe complementary to the substrate RNA or detection of adenosine monophosphate (AMP). The assays are sensitive, flexible, and can be used in low-throughput tube-based formats and 96-well or 384-well plate formats. The assays rely on plate reader detection and can be carried out using manual pipetting or robotic liquid handling equipment. In addition to two activity assays, we describe differential scanning fluorimetry (thermal shift assay) as a complementary assay that allows the direct characterization of ligand binding to deadenylase enzymes. The assays can be useful for the characterization of deadenylase variants and are particularly suitable for the discovery and development of small-molecule inhibitors of deadenylase enzymes.

Automated high-throughput, high-content 3D imaging of intact pancreatic islets.

Diabetes poses a global health crisis affecting individuals across age groups and backgrounds, with a prevalence estimate of 700 million people worldwide by 2045. Current therapeutic strategies primarily rely on insulin therapy or hypoglycemic agents, which fail to address the root cause of the disease - the loss of pancreatic insulin-producing beta-cells. Therefore, bioassays that recapitulate intact islets are needed to enable drug discovery for beta-cell replenishment, protection from beta-cell loss, and islet-cell interactions. Standard cancer insulinoma beta-cell lines MIN6 and INS-1 have been used to interrogate beta-cell metabolic pathways and function but are not suitable for studying proliferative effects. Screening using primary human/rodent intact islets offers a higher level of physiological relevance to enhance diabetes drug discovery and development. However, the 3-dimensionality of intact islets have presented challenges in developing robust, high-throughput assays to detect beta-cell proliferative effects. Established methods rely on either dissociated islet cells plated in 2D monolayer cultures for imaging or reconstituted pseudo-islets formed in round bottom plates to achieve homogeneity. These approaches have significant limitations due to the islet cell dispersion process. To address these limitations, we have developed a robust, intact ex vivo pancreatic islet bioassay in 384-well format that is capable of detecting diabetes-relevant endpoints including beta-cell proliferation, chemoprotection, and islet spatial morphometrics.

High-Content Screening of Synaptic Density Modulators in Primary Neuronal Cultures.

The synapse, which represents the structural and functional basis of neuronal communication, is one of the first elements affected in several neurodegenerative diseases. To better understand the potential role of gene expression in synapse loss, we developed an original high-content screening (HCS) model capable of quantitatively assessing the impact of gene silencing on synaptic density. Our approach is based on a model of primary neuronal cultures (PNCs) from the neonatal rat hippocampus, whose mature synapses are visualized by the relative localization of the presynaptic protein Synaptophysin with the postsynaptic protein Homer1. The heterogeneity of PNCs and the small sizes of the synaptic structures pose technical challenges associated with the level of automation necessary for HCS studies. We overcame these technical challenges, automated the processes of image analysis and data analysis, and carried out tests under real-world conditions to demonstrate the robustness of the model developed. In this article, we describe the screening of a custom library of 198 shRNAs in PNCs in the 384-well plate format. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture of primary hippocampal rat neurons in 384-well plates Basic Protocol 2: Lentiviral shRNA transduction of primary neuronal culture in 384-well plates Basic Protocol 3: Immunostaining of the neuronal network and synaptic markers in 384-well plates Basic Protocol 4: Image acquisition using a high-throughput reader Basic Protocol 5: Image segmentation and analysis Basic Protocol 6: Synaptic density analysis.

Greening automation: Wash and Re-use of disposable 384-well liquid handling tips to enable sustainable high-throughput vaccine development.

Laboratory automation uses large amounts of plastic consumables, generating substantial single-use plastic waste. Automated ELISAs are an indispensable analytical tool in vaccine formulation and process development. Current workflows, however, rely on disposable liquid handling tips. In progress toward sustainability, we developed workflows for washing 384-well format liquid handling tips, using nontoxic reagents, for re-use during ELISA testing. We estimate that this workflow reduces plastic and cardboard waste in our facility by 989kg/year and 202kg/year, respectively, without introducing new chemicals into our waste steam.

A Human Neuron/Astrocyte Co-culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation.

Communication and contact between neurons and astrocytes is important for proper brain physiology. How neuron/astrocyte crosstalk is affected by intraneuronal tau aggregation in neurodegenerative tauopathies is largely elusive. Human induced pluripotent stem cell (iPSC)-derived neurons provide the opportunity to model tau pathology in a translationally relevant in vitro context. However, current iPSC models inefficiently develop tau aggregates, and co-culture models of tau pathology have thus far utilized rodent astrocytes. In this article, we describe the co-culture of human iPSC-derived neurons with primary human astrocytes in a 96-well format compatible with high-content microscopy. By lentiviral overexpression of different mutated tau variants, this protocol can be flexibly adapted for the efficient induction of seeded or spontaneous tau aggregation. We used this novel co-culture model to identify cell type-specific disease mechanisms and to provide proof of concept for intervention by antisense therapy. These results show that this human co-culture model provides a highly translational tool for target discovery and drug development for human tauopathies. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Human neuron/astrocyte co-culture for seeded and spontaneous intraneuronal tau aggregation Support Protocol 1: Human induced pluripotent stem cell culture Support Protocol 2: Human primary astrocyte culture.

Three-Dimensional Bioprinting of Human iPSC-Derived Neuron-Astrocyte Cocultures for Screening Applications.

For a cell model to be viable for drug screening, the system must meet throughput and homogeneity requirements alongside having an efficient development time. However, many published 3D modelsdo not satisfy these criteria. This therefore, limits their usefulness in early drug discovery applications. Three-dimensional (3D) bioprinting is a novel technology that can be applied to the development of 3D models to expedite development time, increase standardization, and increase throughput. Here, we present a protocol to develop 3D bioprinted coculture models of human induced pluripotent stem cell (iPSC)-derived glutamatergic neurons and astrocytes. These cocultures are embedded within a hydrogel matrix of bioactive peptides, full-length extracellular matrix (ECM) proteins, and with a physiological stiffness of 1.1 kPa. The model can be rapidly established in 96-well and 384-well formats and produces an average post-print viability of 72%. The astrocyte-to-neuron ratio in this model is shown to be 1:1.5, which is within the physiological range for the human brain. These 3D bioprinted cell populations also show expression of mature neural cell type markers and growth of neurite and astrocyte projections within 7 days of culture. As a result, this model is suitable for analysis using cell dyes and immunostaining techniques alongside neurite outgrowth assays. The ability to produce these physiologically representative models at scale makes them ideal for use in medium-to-high throughput screening assays for neuroscience targets.

Integrated High-Throughput Screening and Large-Scale Isobolographic Analysis to Accelerate the Discovery of Radiosensitizers With Greater Selectivity for Cancer Cells

High-throughput screening (HTS) platforms have been widely used to identify candidate anticancer drugs and drug-drug combinations; however, HTS-based identification of new drug-ionizing radiation (IR) combinations has rarely been reported. Herein, we developed an integrated approach including cell-based HTS and computational large-scale isobolographic analysis to accelerate the identification of radiosensitizing compounds acting strongly and more specifically on cancer cells. In a 384-well plate format, 160 compounds likely to interfere with the cell response to radiation were screened on human glioblastoma (U251-MG) and cervix carcinoma (ME-180) cell lines, as well as on normal fibroblasts (CCD-19Lu). After drug exposure, cells were irradiated or not and short-term cell survival was assessed by high-throughput cell microscopy. Computational large-scale dose-response and isobolographic approach were used to identify promising synergistic drugs radiosensitizing cancer cells rather than normal cells. Synergy of a promising compound was confirmed on ME-180 cells by an independent 96-well assay protocol, and finally, by the gold-standard colony forming assay. We retained 4 compounds synergistic at 2 isoeffects in U251-MG and ME-180 cell lines and 11 compounds synergistically effective in only one cancer cell line. Among these 15 promising radiosensitizers, 5 compounds showed limited toxicity combined or not with IR on normal fibroblasts. Overall, this study demonstrated that HTS chemoradiation screening together with large-scale computational analysis is an efficient tool to identify synergistic drug-IR combinations, with concomitant assessment of unwanted toxicity on normal fibroblasts. It sparks expectations to accelerate the discovery of highly desired agents improving the therapeutic index of radiation therapy.

Abstract PO-077: High-throughput 3D-spheroid invasion assay: A powerful tool to identify novel drugs targeting tumor micro-environment in HNSCC

Abstract Cancer metastasis is a complex cascade that involves activation of cancer cell invasion and migration. This activation is greatly attributed by multiple factors, including the tumor-microenvironment (TME). Cancer-associated fibroblasts (CAF), a prominent cell type within the TME, have been shown to promote cancer cell invasion and migration, causing metastasis in various cancers including head and neck squamous cell carcinoma (HNSCC). The molecular mechanisms of how CAFs promote HNSCC invasion remain elusive. Our research goal is to understand how CAFs influence HNSCC cells to become invasive and potentially metastatic. Using a top-down research approach, our aim is to identify novel therapeutic chemical probe/drug regimens that potentially target CAF-dependent HNSCC cancer cell-invasion. In-order to perform high-throughput small molecule screens, we have established a 384-well format, three-dimensional (3D) spheroid invasion assay, as a powerful tool to study CAF-dependent HNSCC cancer cell invasion. This platform is currently being used to screen small molecule libraries and identify putative molecular targets, providing insights into underlying mechanisms of CAF-induced cancer cell invasion and candidate therapeutic strategies. Citation Format: Kunal Karve, Stephanie Poon, Panagiotis Prinos, Laurie Ailles. High-throughput 3D-spheroid invasion assay: A powerful tool to identify novel drugs targeting tumor micro-environment in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-077.

High-throughput quantification of emerging ''nitazene'' benzimidazole opioid analogs by microextraction and UHPLC-MS/MS.

Benzimidazole opioids, often referred to as nitazenes, represent a subgroup of new psychoactive substances with a recent increase in fatal overdoses in the US and Europe. With a variety of analogs emerging on the illicit drug market, forensic labs are challenged to identify these potent drugs. We here present a simple quantitative approach for the determination of nine nitazene analogs, namely, clonitazene, etodesnitazene, etonitazene, etonitazepyne, flunitazene, isotonitazene, metodesnitazene, metonitazene, and protonitazene in whole blood using liquid-phase microextraction and electromembrane extraction in a 96-well format and liquid chromatography tandem mass spectrometry. Green and efficient sample preparation was accomplished by liquid-phase microextraction in a 96-well format and resulted in high extraction yields for all analytes (> 81%). Here, blood diluted with buffer (1:1, %v) was extracted from a donor compartment across a thin organic liquid membrane and into an aqueous acceptor solution. The acceptor solution was collected and directly injected into the analysis platform. Chromatographic separation was accomplished with a biphenyl column, allowing for baseline separation of the structural isomers isotonitazene and protonitazene before detection by multiple reaction monitoring. The validation was performed according to Scientific Working Group of Forensic Toxicology guidelines. The calibration range was from 0.5 to 50 nM (except for protonitazene and clonitazene from 0.1 nM) with good linearity and limits of detection down to 0.01 nM. An AGREEprep assessment was performed to evaluate sample preparation greenness, with a final score of 0.71. Nitazenes represent a current threat to public health, and analytical methods that cover a wide range of these analogs are limited. The here described method may assist in the detection of nitazenes in whole blood and prevent these substances from being missed in post-mortem investigations.

РЕГУЛЯЦИЯ СИСТЕМНОГО ВОСПАЛИТЕЛЬНОГО ОТВЕТА У ПАЦИЕНТОВ С МУЛЬТИФОКАЛЬНЫМ АТЕРОСКЛЕРОЗОМ

Introduction. Data on the role of inflammation in the progression and manifestation of multifocal atherosclerosis (MFA) are contradictory. The systemic process of multiple lesions of different vascular beds in patients with an established diagnosis of coronary heart disease (CHD) is due to several parallel pathological mechanisms. In general, in the pathogenesis of both coronary atherosclerosis and atherosclerosis of peripheral arteries, one of the factors is the variability of immune response genes and cytokines, regulators of the inflammatory response. The aim of this work was to determine the role of variability in the genes of cytokines and innate immune response receptors in the structure of mechanisms associated with the development of MFA in patients with coronary artery atherosclerosis. Materials and methods. 260 patients with CAD (mean age 58 years) were examined. Of all those included in the study, at the time of the survey, MFA was found in 180 people (69.23%), while the incidence of MFA in men and women is comparable (70.33 vs. 64.71%, respectively). There is also no significant difference in the incidence of MFA in the two age groups (up to 60 years, 45% have lesions of two or more vascular beds, and in people older than 60 years, MFA was found in 55% of cases). All patients were informed about the course of the study and voluntarily signed an informed consent. Blood was collected from the cubital vein into Vacutainer tubes with K3EDTA. DNA isolation was carried out using the phenol-chloroform extraction method. Genotyping of 47 polymorphic sites with a single nucleotide change (SNV) of 19 genes proven to be associated with the regulation of the systemic inflammatory response (cytokines, innate immunity receptors) was performed in a 96-well format using TaqMan technology (LifeTechnologies, USA) and real-time result detection with using the ViiATM 7 RealTime PCR System (LifeTechnologies, USA) for polymerase chain reaction in accordance with the instructions. Associations were analyzed using Snpstats (https://www.snpstats.net/start.htm). Odds ratio (OR) and 95% CI for OR were calculated for risk assessment. Results. One association has been established that demonstrates a negative impact and increases the risk of developing MFA in young and middle age. Carriage of the G rs11685424 IL1RL1 allele increases the risks of earlier development of peripheral arterial lesions by 2 times (OR 2.11 (95% CI 1.09-4.06), p = 0.024) according to the recessive inheritance model. At the same time, variability in three sites (rs1974675; rs6758936; rs3755276) of the IL18 receptor gene (IL18R1) reduces the risk of MFA detection by three times (p=0.12; p=0.034: p=0.026, respectively) and this pattern is also observed in patients older age group. In the carriers of the A/A rs1974675, T/T rs3755276 and A/A rs6758936 genotypes in persons over 60 years of age, MFA was diagnosed less frequently (p=0.024; p=0.037; p=0.011, respectively). Conclusion. It has been shown that in patients with significant coronary atherosclerosis, the progression of peripheral vascular atherosclerosis is more associated with the variability of the genes responsible for the regulation of the systemic inflammatory response, rather than genes and their variants that determine the severity and strength of the immune response.

Development and Validation of a HTS Platform for the Discovery of New Antifungal Agents against Four Relevant Fungal Phytopathogens.

Fungal phytopathogens are the major agents responsible for causing severe damage to and losses in agricultural crops worldwide. Botrytis cinerea, Colletotrichum acutatum, Fusarium proliferatum, and Magnaporthe grisea are included in the top ten fungal phytopathogens that impose important plant diseases on a broad range of crops. Microbial natural products can be an attractive alternative for the biological control of phytopathogens. The objective of this work was to develop and validate a High-throughput Screening (HTS) platform to evaluate the antifungal potential of chemicals and natural products against these four important plant pathogens. Several experiments were performed to establish the optimal assay conditions that provide the best reproducibility and robustness. For this purpose, we have evaluated two media formulations (SDB and RPMI-1640), several inoculum concentrations (1 × 106, 5 × 105 and 5 × 106 conidia/mL), the germination curves for each strain, each strain's tolerance to dimethyl sulfoxide (DMSO), and the Dose Response Curves (DRC) of the antifungal control (Amphotericin B). The assays were performed in 96-well plate format, where absorbance at 620 nm was measured before and after incubation to evaluate growth inhibition, and fluorescence intensity at 570 nm excitation and 615 nm emission was monitored after resazurin addition for cell viability evaluation. Quality control parameters (RZ' Factors and Signal to Background (S/B) ratios) were determined for each assay batch. The assay conditions were finally validated by titrating 40 known relevant antifungal agents and testing 2400 microbial natural product extracts from the MEDINA Library through both HTS agar-based and HTS microdilution-based set-ups on the four phytopathogens.

Temporal modelling of the biofilm lifecycle (TMBL) establishes kinetic analysis of plate-based bacterial biofilm dynamics

Bacterial biofilms are critical to pathogenesis and infection. They are associated with rising rates of antimicrobial resistance. Biofilms are correlated with worse clinical outcomes, making them important to infectious diseases research. There is a gap in knowledge surrounding biofilm kinetics and dynamics which makes biofilm research difficult to translate from bench to bedside. To address this gap, this work employs a well-characterized crystal violet biomass accrual and planktonic cell density assay across a clinically relevant time course and expands statistical analysis to include kinetic information in a protocol termed the TMBL (Temporal Mapping of the Biofilm Lifecycle) assay. TMBL's statistical framework quantitatively compares biofilm communities across time, species, and media conditions in a 96-well format. Measurements from TMBL can reliably be condensed into response features that inform the time-dependent behavior of adherent biomass and planktonic cell populations. Staphylococcus aureus and Pseudomonas aeruginosa biofilms were grown in conditions of metal starvation in nutrient-variable media to demonstrate the rigor and translational potential of this strategy. Significant differences in single-species biofilm formation are seen in metal-deplete conditions as compared to their controls which is consistent with the consensus literature on nutritional immunity that metal availability drives transcriptomic and metabolomic changes in numerous pathogens. Taken together, these results suggest that kinetic analysis of biofilm by TMBL represents a statistically and biologically rigorous approach to studying the biofilm lifecycle as a time-dependent process. In addition to current methods to study the impact of microbe and environmental factors on the biofilm lifecycle, this kinetic assay can inform biological discovery in biofilm formation and maintenance.

Development of a membrane-based Gi-CASE biosensor assay for profiling compounds at cannabinoid receptors.

Introduction: The cannabinoid receptor (CBR) subtypes 1 (CB1R) and 2 (CB2R) are key components of the endocannabinoid system (ECS), playing a central role in the control of peripheral pain, inflammation and the immune response, with further roles in the endocrine regulation of food intake and energy balance. So far, few medicines targeting these receptors have reached the clinic, suggesting that a better understanding of the receptor signalling properties of existing tool compounds and clinical candidates may open the door to the development of more effective and safer treatments. Both CB1R and CB2R are Gαi protein-coupled receptors but detecting Gαi protein signalling activity reliably and reproducibly is challenging. This is due to the inherent variability in live cell-based assays and restrictions around the use of radioactive [35S]-GTPγS, a favoured technology for developing higher-throughput membrane-based Gαi protein activity assays. Methods: Here, we describe the development of a membrane-based Gαi signalling system, produced from membrane preparations of HEK293TR cells, stably overexpressing CB1R or CB2R, and components of the Gαi-CASE biosensor. This BRET-based system allows direct detection of Gαi signalling in both cells and membranes by monitoring bioluminescence resonance energy transfer (BRET) between the α and the βγ subunits. Cells and membranes were subject to increasing concentrations of reference cannabinoid compounds, with 10μM furimazine added to generate RET signals, which were detected on a PHERAstar FSX plate reader, then processed using MARS software and analysed in GraphPad PRISM 9.2. Results: In membranes expressing the Gi-CASE biosensor, the cannabinoid ligands profiled were found to show agonist and inverse agonist activity. Agonist activity elicited a decrease in the BRET signal, indicative of receptor activation and G protein dissociation. Inverse agonist activity caused an increase in BRET signal, indicative of receptor inactivation, and the accumulation of inactive G protein. Our membrane-based Gi-CASE NanoBRET system successfully characterised the potency (pEC50) and efficacy (Emax) of CBR agonists and inverse agonists in a 384-well screening format. Values obtained were in-line with whole-cell Gi-CASE assays and consistent with literature values obtained in the GTPγS screening format. Discussion: This novel, membrane-based Gαi protein activation assay is applicable to other Gαi-coupled GPCRs, including orphan receptors, allowing real-time higher-throughput measurements of receptor activation.

Human skin stem cell-derived hepatic cells as in vitro drug discovery model for insulin-driven de novo lipogenesis

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is characterized by intrahepatic triglyceride accumulation and can progress to metabolic dysfunction-associated steatohepatitis (MASH) and liver fibrosis. Hepatic de novo lipogenesis (DNL), activated by glucose and insulin, is a central pathway contributing to early-stage development of MASLD. The emerging global prevalence of MASLD highlights the urgent need for pharmaceutical intervention to combat this health threat. However, the identification of novel drugs that could inhibit hepatic DNL is hampered by a lack of reliable, insulin-sensitive, human, in vitro, hepatic models. Here, we report human skin stem cell-derived hepatic cells (hSKP-HPC) as a unique in vitro model to study insulin-driven DNL (iDNL), evidenced by both gene expression and lipid accumulation readouts. Insulin-sensitive hSKP-HPC showed increased sterol regulatory element-binding protein 1c (SREBP-1c) expression, a key transcription factor for DNL. Furthermore, this physiologically relevant in vitro human steatosis model allowed both inhibition and activation of the iDNL pathway using reference inhibitors and activators, respectively. Optimisation of the lipid accumulation assay to a high-throughput, 384-well format enabled the screening of a library of annotated compounds, delivering new insights on key players in the iDNL pathway and MASLD pathophysiology. Together, these results establish the value of the hSKP-HPC model in preclinical development of antisteatotic drugs to combat MASLD.

Evaluation of a high-throughput H295R homogenous time resolved fluorescence assay for androgen and estrogen steroidogenesis screening

The H295R test guideline assay evaluates the effect of test substances on synthesis of 17β-estradiol (E2) and testosterone (T). The objective of this study was to leverage commercial immunoassay technology to develop a more efficient H295R assay to measure E2 and T levels in 384-well format. The resulting Homogenous Time Resolved Fluorescence assay platform (H295R-HTRF) was evaluated against a training set of 36 chemicals derived from the OECD inter-laboratory validation study, EPA guideline 890.1200 aromatase assay, and azole fungicides active in the HT-H295R assay. Quality control performance criteria were met for all conditions except E2 synthesis inhibition where low basal hormone synthesis was observed. Five proficiency chemicals were active for both the E2 and T endpoints, consistent with guideline classifications. Of the nine OECD core reference chemicals, 9/9 were concordant with outcomes for E2 and 7/9 for T. Likewise, 9/13 and 11/13 OECD supplemental chemicals were concordant with anticipated effects for E2 and T, respectively. Of the 10 azole fungicides screened, 7/10 for E2 and 8/10 for T exhibited concordant outcomes for inhibition. Generally, all active chemicals in the training set demonstrated equivalent or greater potency in the H295R-HTRF assay, supporting the sensitivity of the platform. The adaptation of HTRF technology to the H295R model provides an efficient way to evaluate E2 and T modulators in accordance with guideline specifications.

Abstract P1034: Functional Modelling Of Arrhythmias With Human Cardiac Organoids

Engineered heart tissues (EHTs) have become a well-established model of human cardiac muscle. Metabolic maturity, improved sarcoplasmic reticulum (SR) Ca2+ handling, and the incorporation of critical non-myocyte cell populations are advantages that EHTs provide over immature 2D cardiomyocyte models. Our human cardiac organoid-EHT hybrid (hCO) platform was developed to be compatible with standardized 96-well tissue culture formats to enable high-throughput functional screening, while retaining the physiological advantages of larger EHTs. Here, we explored the applicability of hCOs as a model of various arrhythmic mechanisms. QT-prolongation could be mimicked with LTCC agonist BayK8644 or hERG blocker dofetilide, which increased the relaxation time of the hCOs and induced arrhythmia at high concentrations. Ectopy was induced by perturbing SR Ca2+ cycling with ryanodine or caffeine. HCN4 blocker ivabradine slowed rate, and at high concentrations produced a complex pause-burst phenotype. Increasing the cardiomyocyte-to-fibroblast ratio with growth factors, or the addition of extra fibroblasts prolonged contraction duration and induced ectopy. Inflammatory cytokines also increased relaxation time and provoked ectopy. From this data we propose that hCOs are a sensitive and reliable platform for studying arrhythmias and we are currently exploring the impact of CPVT-linked RYR2 and CASQ2 mutations on hCO arrhythmogenesis.

Abstract P2169: Identification Of Pro-adhesive Drugs By A Novel High-throughput Approach

Defective cell-cell adhesion contributes to the patho-mechanism of various diseases. In line with this, a mouse model recently developed by our group demonstrate that loss of cell-cell adhesion is an important initial step leading to Arrhythmogenic Cardiomyopathy (ACM), a disease which presents with severe arrhythmia, ventricular fibrosis and impaired cardiac function. Derived from the central role of defective cell-cell adhesion for the patho-mechanism, we here aim to identify new compounds, which restore intercellular adhesion and could serve as potential therapeutics. To screen a high number of agents, we developed an adhesion-based high-throughput approach by adapting a standard cell-cell dissociation assay to 96-well plate format including establishment of an automated acquisition and analysis pipeline. To model loss of intercellular adhesion, cells deficient for the desmosomal adhesion molecule desmoglein-2 were used. After establishing settings to sensitively detect changes in cell-cell adhesion, this method was applied to screen a library of 1946 FDA-approved drugs in three concentrations. Several new compounds strengthening intercellular adhesion were identified in addition to known pro-adhesive drugs, confirming the robustness of our assay. The increased adhesiveness of selected top hits was confirmed in cells expressing ACM patient mutations. In conclusion, we here developed an adhesion-based high-throughput approach capable of identifying adhesion modulators which have the potential to be used as therapeutics for diseases with defective desmosomal adhesion such as ACM.

Automated and robust extraction of genomic DNA from various leftover blood samples

With the development of genomic technologies, the isolation of genomic DNA (gDNA) from clinical samples is increasingly required for clinical diagnostics and research studies. In this study, we explored the potential of utilizing various leftover blood samples obtained from routine clinical tests as a viable source of gDNA. Using an automated method with optimized pre-treatments, we obtained gDNA from seven types of clinical leftover blood, with average yields of gDNA ranging from 3.11 ± 0.45 to 22.45 ± 4.83 μg. Additionally, we investigated the impact of storage conditions on gDNA recovery, resulting in yields of 8.62–68.08 μg when extracting gDNA from EDTA leftover blood samples stored at 4 °C for up to 13 weeks or −80 °C for up to 78 weeks. Furthermore, we successfully obtained sequenceable gDNA from both Serum Separator Tube and EDTA Tube using a 96-well format extraction, with yields ranging from 0.61 to 71.29 μg and 3.94–215.98 μg, respectively. Our findings demonstrate the feasibility of using automated high-throughput platforms for gDNA extraction from various clinical leftover blood samples with the proper pre-treatments.

Validated LC-MS/MS method for simultaneous quantification of KRASG12C inhibitor sotorasib and its major circulating metabolite (M24) in mouse matrices and its application in a mouse pharmacokinetic study

We have successfully developed and validated a bioanalytical assay using liquid chromatography tandem mass spectrometry to simultaneously quantify the first approved KRASG12C inhibitor sotorasib and its major circulating metabolite (M24) in various mouse matrices. M24 was synthesized in-house via low-pH hydrolysis. We utilized a fast and efficient protein precipitation method in a 96-well plate format to extract both analytes from biological matrices. Erlotinib was selected as the internal standard in this assay. Gradient elution using methanol and 0.1 % formic acid in water (v/v) was applied on an Acquity UPLC BEH C18 column to separate all analytes. Sotorasib, M24, and erlotinib were detected with a triple quadrupole mass spectrometer in positive electrospray ionization in multiple reaction monitoring mode. During the validation and sample quantification, a linear calibration range was observed for both sotorasib and M24 in a range of 4 – 4000 nM and 1 – 1000 nM, respectively. The %bias and %CV (both intra- and inter-day) for all tested levels in all investigated matrices were lower than 15 % as required by the guidelines. Sotorasib had a rather short room temperature stability in mouse plasma for up to 8 h compared to M24 which was stable up to 16 h at room temperature. This method has been successfully applied to measure sotorasib and M24 in several mouse matrices from three different mouse strains. We can conclude that the plasma exposure of sotorasib in mice is limited via human CYP3A4- and mouse Cyp3a-mediated metabolism of sotorasib into M24.

Characterization of neurotransmitter inhibition for seven cathinones by a proprietary fluorescent dye method.

Many new psychoactive substances (NPS) are stimulants, and information about their potency and abuse potential is often lacking. To start addressing this need, a method measuring the inhibition of the dopamine, serotonin, and norepinephrine transporters (DAT, SERT, and NET) by stimulant drugs was developed. The use of a proprietary fluorescent dye mixture and three cell lines (CHO-K1, HEK 293, and MDCK), each expressing a single transporter, allowed for a semiautomated, one-pot determination of inhibition in a 384-well format. The method was validated using well characterized stimulants, including cocaine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), α-PVP, and fluoxetine and performed similarly to other methods. Seven synthetic cathinones all showed highest potency for DAT inhibition, followed by NET and SERT. The rank potency for DAT inhibition IC50 (nM) was MPHP (4.53) > 4Cl-α-PVP (8.05) > 3F-α-PVP (12.7) > α-PiHP (13.4) > N-ethylpentylone (16.9) > N-ethylhexedrone (44.5) > 4-methylpentedrone (261). All but 4-methylpentedrone were more potent than amphetamine (257) and cocaine (111). The DAT/SERT inhibition ratio for the cathinones was in the range from 5.02 for 4-methylpentedrone to >3730 for α-PiHP, compared to 1.64 for cocaine and >4030 for α-PVP. All seven substances had inhibition profiles similar to those of potent stimulants with high abuse potential.