High-throughput Screening Format Research Articles

Abstract 7178: Neuronal cells derived from iPSCs cell to evaluate neurotoxicity after 48 or 72 hours in high-through put screening format

Abstract Neurotoxicity is a major concern in central nervous system drug discovery and a frequent cause of attrition in clinical trials or approved drug withdrawal. Neuropathy is a side effect defined by the injury of peripheral nerves causing a loss of sensation/motion. Drug development pipelines typically involve testing in cell lines followed by animal investigations with translation to humans. The induced pluripotent stem cells (iPSCs) have widened new model systems to study adverse toxicities. With evolution in regulatory guidance the use of human- iPSC-derived tissue provides high-throughput access and a more relevant testing environment. Neuropathy is one of the most chemotherapy adverse effects. In this study we have screened 8 compounds. Bortezomib (proteasome inhibitor), gemcitabine (approved ovarian cancer treatment), ivermectin (parasitic diseases treatment), flavopiridol (approved CDKs inhibitors), SNS-032 (approved CDKs inhibitor), mefloquine (anti-malaria drug), digitonin (nonionic detergent) and tamoxifen (hormone receptor-positive breast cancer treatment). These molecule toxicities were tested on neuronal-derived iPSCs Dopaneurone, GABAneurone, Glutaneurone, Motorneurone, Microglya and Astrocytes in high throughput screening. For the project, cells were plated in 384 well/plates. DMSO tolerance was performed (up to 10%). Drugs were added, then, after 48h or 72h, total protease and protease activities were measured using CytoTox-GloTM. Compounds were tested at 7 concentrations (from 10−4 to 10−9M).In this study we have validated cell culture and the assay conditions. The signal window range was good after 48h and 72h of treatment. No toxicity was found until 10% DMSO. A specific neurotoxicity signature was measurable for each drug tested. Digitonin, our positive control, induced toxicity in all IPSCs after 48h and 72h of incubation. On the contrary, Tamoxifen, our negative control, did not induce any neurotoxicity. The other molecules generated a signature of severe to mild neurotoxicity. These results are in agreement with their known adverse side-effects. In conclusion, this panel is a good tool to anticipate possible neurotoxicity within the 3Rs respect. It can be used in early drug de-risking or neuroprotection screening, with the aim of preventing/reducing/curing neuropathy in at-risk populations. Citation Format: Fabienne Sabatier, Justine Chaigne, Maryse Martin, Rodolphe Barbeau, Luisa Houssin, Stephane Bonnin, Alastair King, Vanessa Jahnke. Neuronal cells derived from iPSCs cell to evaluate neurotoxicity after 48 or 72 hours in high-through put screening format [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7178.

Abstract 221: Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies

Abstract Organotypic, three-dimensional (3D) cancer tissue models (OCTM) have enabled investigators to develop predictive markers that can be used in preclinical cancer drug development. However, a drawback of the existing models is that they are mostly based on cell lines based are not physiological. In vitro development of 3D tumoroids using patient derived primary tumors (PDPT) in tumor microenvironment (TME) will have clinical significant and can predict human responses to cancer therapeutics. Here, we describe the generation of OCTM from patient-derived metastatic colorectal cancer, the second most deadly cancer. PDPT were grown in Matrigel for 5 passages and for biobanking purposes tumoroids were further expanded in faCellitate 384 well plates or Sun Bioscience plates to harvest large amounts of well-defined tumoroids cultured for over 21 days. Expanded tumoroids were collected and cryopreserved or gently mixed several times using a pipet tip to break up tumoroids into smaller fragments. These tumor fragments were mixed with endothelial cells, dendritic cells, fibroblasts and embedded in a collagen matrix to simulate TME. The collagen-cell mixture was then added to Transwells and induced to gel to form a collagen gel matrix that simulates TME. The tumoroids were cultured with a specialized medium that supports the growth of complex cell types. The tumor development was monitored daily using bright field microscopy and image analysis was used to monitor tumor size. To evaluate the utility of the OCTM, treatment was initiated at day 10 of the culture period by exposing tissues with the chemotherapeutic drug Cisplatin on days 0, 2, 4, 7, 9, and 11. After each treatment, OCTM tissues (N=2) were fixed for H&E and live/dead staining. Overall, the results of the study showed that: 1) PDPT can be expanded for 5 passages (this low passage number is not expected to change the phenotype of the tumor) and further expanded in high throughput format, 384 well plates, 2) the PDPT could be cultured with multiple cell types in a collagen-gel matrix to recreate the TME, 3) histological evaluation of the OCTM showed a glandular-like tumoroid, 4) immunohistochemical staining revealed that the PDPT in the in vivo-like microenvironment were positive for CK20, and 5) the PDPT shrank following 3 treatment cycles with Cisplatin (day 4). Conclusion: The data shows metastatic colorectal PDPT can be expanded in vitro and cultured in a complex tumor microenvironment on tissue culture inserts. These OCTMs can be used to screen drug safety and efficacy. This study opens new avenues for high-throughput cancer drug screening format using PDPT embedded in a in vivo like TME. This model can be used as a tool for personalized oncology drug testing and for translational assays tailored to other cancer types from our patient derived primary tumor pool. Citation Format: Seyoum Ayehunie, Megan Groves, Bryda Bryda, Alex Armento, Anthony Tolcher. Novel organotypic patient derived primary tumor tissues for oncology drug safety and efficacy studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 221.

Quantitative, titratable and high-throughput reporter assays to measure DNA double strand break repair activity in cells.

Repair of DNA damage is essential for the maintenance of genome stability and cell viability. DNA double strand breaks (DSBs) constitute a toxic class of DNA lesion and multiple cellular pathways exist to mediate their repair. Robust and titratable assays of cellular DSB repair (DSBR) are important to functionally interrogate the integrity and efficiency of these mechanisms in disease models as well as in response to genetic or pharmacological perturbations. Several variants of DSBR reporters are available, however these are often limited by throughput or restricted to specific cellular models. Here, we describe the generation and validation of a suite of extrachromosomal reporter assays that can efficiently measure the major DSBR pathways of homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single strand annealing (SSA). We demonstrate that these assays can be adapted to a high-throughput screening format and that they are sensitive to pharmacological modulation, thus providing mechanistic and quantitative insights into compound potency, selectivity, and on-target specificity. We propose that these reporter assays can serve as tools to dissect the interplay of DSBR pathway networks in cells and will have broad implications for studies of DSBR mechanisms in basic research and drug discovery.

The Novel Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) Platform Identifies Synergistic Activity of Lanraplenib and Ruxolitinib in Hematological Malignancies

Background: Investigation of drug combinations across different contexts can provide useful insights on the anti-cancer mechanism and can ultimately lead to new treatments. However, conventional drug combination screening methods are limited by throughput. Efforts to systematically identify the most effective active combinations and the optimal molecular contexts in a high throughput screening (HTS) format could greatly accelerate the development of combination treatments. Spleen Tyrosine Kinase, SYK, is a non-receptor tyrosine kinase known to regulate intracellular signaling, including FLT3, AKT/mTOR and STAT5 pathways, via its immunoreceptor tyrosine-based activation motif (ITAM). Deregulated SYK signaling has been reported to play a central role in pathogenesis of allergic and autoimmune diseases as well as hematological malignancies. Lanraplenib (LANRA) is a next-generation SYK inhibitor currently being evaluated in combination with gilteritinib, a FLT3 inhibitor, in patients with relapsed or refractory (R/R) FLT3-mutated acute myeloid leukemia (AML) (NCT05028751). Given its critical role in intracellular signaling and interaction with receptor tyrosine kinases(RTKs), we hypothesized that LANRA could synergize with ruxolitinib, a JAK inhibitor. To address this hypothesis, we performed a high throughput drug combination screen using the Broad Institute's Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) platform, which enables rapid screening of thousands of compounds in a 930-cell line panel across 45 different lineages. Methods: In this study, we performed a combination screen using the PRISM platform with ruxolitinib as the test compound, with a 10 μM top dose (7 dose concentrations with 3-fold dilutions), in combination with LANRA at two anchor doses, 2 μM and 10 μM. PRISM is a pooled, multiplexed cell viability assay that provides 7-point dose response curves, IC 50, AUC values, and relative abundance of unique cell line barcodes. To understand the drug synergy landscape across different lineages, we developed a bioinformatics pipeline which uses PRISM viability data to calculate synergy scores across all the cell lines and drug combinations. Secondary validation studies of the combinations used CTG viability measurements. Phospho-SYK expression was evaluated in archival formalin-fixed paraffin-embedded (FFPE) bone marrow biopsies from patients with myeloid proliferative neoplasm (MPN) by immunohistochemistry (IHC). RNA-seq was performed to evaluate differential changes in gene expression in response to LANRA. Gene set enrichment analysis (GSEA) was performed to evaluate perturbation in leukemogenic signaling pathways. Results: In the PRISM cell line panel, ruxolitinib in combination with LANRA demonstrated synergistic activity in hematological malignancy cell lines. Among AML cell lines in the panel, OCIAML5, OCIM1, HL60, HEL, EOL1, MONOMAC6, NB4, U937, PL21, and TF1 showed the highest synergy scores. The most sensitive cell lines to the combination showed up-regulation of JAK-STAT and inflammatory signaling pathways in a gene set enrichment analysis (GSEA) prior to treatment. Consistent with this, phosphorylated SYK was associated with inflammatory megakaryocytes and fibrosis in primary samples from patients with MPN. LANRA down-regulated JAK-STAT signaling in a reporter cell line and repressed gene expression associated with dysregulated inflammatory pathways in AML cells. Additionally, the combination of LANRA and ruxolitinib showed synergistic antiproliferative activity across a broad range of concentrations in FLT3-ITD primary AML cells and a panel of hematological malignancy cell lines, confirming the PRISM results. Conclusion: These studies demonstrate the utility of PRISM as a platform to rapidly identify rational combination agents. Importantly, LANRA is effective in combination with ruxolitinib in AML and other hematological malignancy preclinical models. This finding is consistent with the observation that SYK can regulate STAT signaling and cooperate with other RTKs like FLT3. Given the central role of SYK in regulation of oncogenic and inflammatory signaling, SYK inhibition with LANRA in combination with ruxolitinib may be a promising strategy for patients with myeloid malignancies.

Novel inhibitors that target bacterial virulence identified via HTS against intra-macrophage survival of Shigella flexneri.

Shigella flexneri is a facultative intracellular pathogen that causes shigellosis, a human diarrheal disease characterized by the destruction of the colonic epithelium. Novel antimicrobial compounds to treat infections are urgently needed due to the proliferation of bacterial antibiotic resistance and lack of new effective antimicrobials in the market. Our approach to find compounds that block the Shigella virulence pathway has three potential advantages: (i) resistance development should be minimized due to the lack of growth selection pressure, (ii) no resistance due to environmental antibiotic exposure should be developed since the virulence pathways are not activated outside of host infection, and (iii) the normal intestinal microbiota, which do not have the targeted virulence pathways, should be unharmed. We chose to utilize two phenotypic assays, inhibition of Shigella survival in macrophages and Shigella growth inhibition (minimum inhibitory concentration), to interrogate the 1.7 M compound screening collection subset of the GlaxoSmithKline drug discovery chemical library. A number of secondary assays on the hit compounds resulting from the primary screens were conducted, which, in combination with chemical, structural, and physical property analyses, narrowed the final hit list to 44 promising compounds for further drug discovery efforts. IMPORTANCE The rapid development of antibiotic resistance is a critical problem that has the potential of returning the world to a "pre-antibiotic" type of environment, where millions of people will die from previously treatable infections. One relatively newer approach to minimize the selection pressures for the development of resistance is to target virulence pathways. This is anticipated to eliminate any resistance selection pressure in environmental exposure to virulence-targeted antibiotics and will have the added benefit of not affecting the non-virulent microbiome. This paper describes the development and application of a simple, reproducible, and sensitive assay to interrogate an extensive chemical library in high-throughput screening format for activity against the survival of Shigella flexneri 2457T-nl in THP-1 macrophages. The ability to screen very large numbers of compounds in a reasonable time frame (~1.7 M compounds in ~8 months) distinguishes this assay as a powerful tool in further exploring new compounds with intracellular effect on S. flexneri or other pathogens with similar pathways of pathogenesis. The assay utilizes a luciferase reporter which is extremely rapid, simple, relatively inexpensive, and sensitive and possesses a broad linear range. The assay also utilized THP-1 cells that resemble primary monocytes and macrophages in morphology and differentiation properties. THP-1 cells have advantages over human primary monocytes or macrophages because they are highly plastic and their homogeneous genetic background minimizes the degree of variability in the cell phenotype (1). The intracellular and virulence-targeted selectivity of our methodology, determined via secondary screening, is an enormous advantage. Our main interest focuses on hits that are targeting virulence, and the most promising compounds with adequate physicochemical and drug metabolism and pharmacokinetic (DMPK) properties will be progressed to a suitable in vivo shigellosis model to evaluate the therapeutic potential of this approach. Additionally, compounds that act via a host-directed mechanism could be a promising source for further research given that it would allow a whole new, specific, and controlled approach to the treatment of diseases caused by some pathogenic bacteria.

TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS.

SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past 2 years. This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle. The interaction between host transmembrane serine protease 2 (TMPRSS2) and viral SPIKE protein is an important initial step in SARS-CoV-2 infection, offering an opportunity for therapeutic development of viral entry inhibitors. Here, we report the development of a time-resolved fluorescence/Förster resonance energy transfer (TR-FRET) assay for monitoring the TMPRSS2-SPIKE interaction in lysate from cells co-expressing these proteins. The assay was configured in a 384-well-plate format for high-throughput screening with robust assay performance. To enable large-scale compound screening, we further miniaturized the assay into 1536-well ultrahigh-throughput screening (uHTS) format. A pilot screen demonstrated the utilization of the assay for uHTS. Our optimized TR-FRET uHTS assay provides an enabling platform for expanded screening campaigns to discover new classes of small-molecule inhibitors that target the SPIKE and TMPRSS2 protein-protein interaction.

A human liver organoid screening platform for DILI risk prediction

Drug-induced liver injury (DILI), both intrinsic and idiosyncratic, causes frequent morbidity, mortality, clinical trial failures and post-approval withdrawal. This suggests an unmet need for improved in vitro models for DILI risk prediction that can account for diverse host genetics and other clinical factors. In this study, we evaluated the utility of human liver organoids (HLOs) for high-throughput DILI risk prediction and in an organ-on-chip system. HLOs were derived from 3 separate iPSC lines and benchmarked on two platforms for their ability to model in vitro liver function and identify hepatotoxic compounds using biochemical assays for albumin, ALT, and AST, microscopy-based morphological profiling, and single-cell transcriptomics: 1) HLOs dispersed in 384-well formatted plates and exposed to a library of compounds. 2) HLOs adapted to a liver-on-chip system. 1) Dispersed HLOs derived from the 3 iPSC lines had similar DILI predictive capacity to intact HLOs in a high-throughput screening format allowing for measurable IC50 values of compound cytotoxicity. Distinct morphological differences were observed in cells treated with drugs exerting differing mechanisms of toxicity. 2) On-chip HLOs significantly increased albumin production, CYP450 expression, and ALT/AST release when treated with known DILI drugs compared to dispersed HLOs and primary human hepatocytes. On-chip HLOs were able to predict the synergistic hepatotoxicity of tenofovir-inarigivir and showed steatosis and mitochondrial perturbation via phenotypic and transcriptomic analysis with exposure to FIAU and acetaminophen, respectively. The high throughput and liver-on-chip system exhibit enhanced in vivo-like function and demonstrate the potential utility of these platforms for hepatotoxicity risk assessment. Tenofovir-inarigivr associated hepatotoxicity was observed and correlates with the clinical manifestation of DILI observed in patients.

Lumit: A Homogeneous Bioluminescent Immunoassay for Detecting Diverse Analytes and Intracellular Protein Targets.

Traditional immunoassays to detect secreted or intracellular proteins can be tedious, require multiple washing steps, and are not easily adaptable to a high-throughput screening (HTS) format. To overcome these limitations, we developed Lumit, a novel immunoassay approach that combines bioluminescent enzyme subunit complementation technology and immunodetection. This bioluminescent immunoassay does not require washes or liquid transfers and takes less than 2h to complete in a homogeneous "Add and Read" format. In this chapter, we describe step-by-step protocols to create Lumit immunoassays for the detection of (1) secreted cytokines from cells, (2) phosphorylation levels of a specific signaling pathway node protein, and (3) a biochemical protein-protein interaction between a viral surface protein and its human receptor.

A High Throughput Screen with a Clonogenic Endpoint to Identify Radiation Modulators of Cancer.

Clonogenic assays evaluate the ability of single cells to proliferate and form colonies. This process approximates the regrowth and recurrence of tumors after treatment with radiation or chemotherapy, and thereby provides a drug discovery platform for compounds that block this process. However, because of their labor-intensive and cumbersome nature, adapting canonical clonogenic assays for high throughput screening (HTS) has been challenging. We overcame these barriers by developing an integrated system that automates cell- and liquid-handling, irradiation, dosimetry, drug administration, and incubation. Further, we developed a fluorescent live-cell based automated colony scoring methodology that identifies and counts colonies precisely based upon actual nuclei number rather than colony area, thereby eliminating errors in colony counts caused by radiation induced changes in colony morphology. We identified 13 cell lines from 7 cancer types, where radiation is a standard treatment module, that exhibit identical radiation and chemoradiation response regardless of well format and are amenable to miniaturization into small-well HTS formats. We performed pilot screens through a 1,584 compound NCI Diversity Set library using two cell lines representing different cancer indications. Radiation modulators identified in the pilot screens were validated in traditional clonogenic assays, providing proof-of-concept for the screen. The integrated methodology, hereafter "clonogenic HTS", exhibits excellent robustness (Z' values > 0.5) and shows high reproducibility (>95%). We propose that clonogenic HTS we developed can function as a drug discovery platform to identify compounds that inhibit tumor regrowth following radiation therapy, to identify new efficacious pair-wise combinations of known oncologic therapies, or to identify novel modulators ofapproved therapies.

A Large-Scale High-Throughput Screen for Modulators of SERCA Activity

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is a P-type ion pump that transports Ca2+ from the cytosol into the endoplasmic/sarcoplasmic reticulum (ER/SR) in most mammalian cells. It is critically important in muscle, facilitating relaxation and enabling subsequent contraction. Increasing SERCA expression or specific activity can alleviate muscle dysfunction, most notably in the heart, and we seek to develop small-molecule drug candidates that activate SERCA. Therefore, we adapted an NADH-coupled assay, measuring Ca-dependent ATPase activity of SERCA, to high-throughput screening (HTS) format, and screened a 46,000-compound library of diverse chemical scaffolds. This HTS platform yielded numerous hits that reproducibly alter SERCA Ca-ATPase activity, with few false positives. The top 19 activating hits were further tested for effects on both Ca-ATPase and Ca2+ transport, in both cardiac and skeletal SR. Nearly all hits increased Ca2+ uptake in both cardiac and skeletal SR, with some showing isoform specificity. Furthermore, dual analysis of both activities identified compounds with a range of effects on Ca2+-uptake and ATPase, which fit into distinct classifications. Further study will be needed to identify which classifications are best suited for therapeutic use. These results reinforce the need for robust secondary assays and criteria for selection of lead compounds, before undergoing HTS on a larger scale.

Prediction of drug-induced liver injury and cardiotoxicity using chemical structure and in vitro assay data

Drug-induced liver injury (DILI) and cardiotoxicity (DICT) are major adverse effects triggered by many clinically important drugs. To provide an alternative to in vivo toxicity testing, the U.S. Tox21 consortium has screened a collection of ∼10K compounds, including drugs in clinical use, against >70 cell-based assays in a quantitative high-throughput screening (qHTS) format. In this study, we compiled reference compound lists for DILI and DICT and compared the potential of Tox21 assay data with chemical structure information in building prediction models for human in vivo hepatotoxicity and cardiotoxicity. Models were built with four different machine learning algorithms (e.g., Random Forest, Naïve Bayes, eXtreme Gradient Boosting, and Support Vector Machine) and model performance was evaluated by calculating the area under the receiver operating characteristic curve (AUC-ROC). Chemical structure-based models showed reasonable predictive power for DILI (best AUC-ROC = 0.75 ± 0.03) and DICT (best AUC-ROC = 0.83 ± 0.03), while Tox21 assay data alone only showed better than random performance. DILI and DICT prediction models built using a combination of assay data and chemical structure information did not have a positive impact on model performance. The suboptimal predictive performance of the assay data is likely due to insufficient coverage of an adequately predictive number of toxicity mechanisms. The Tox21 consortium is currently expanding coverage of biological response space with additional assays that probe toxicologically important targets and under-represented pathways that may improve the prediction of in vivo toxicity such as DILI and DICT.

Katacine Is a New Ligand of CLEC-2 that Acts as a Platelet Agonist.

Background CLEC-2 is a platelet receptor with an important role in thromboinflammation but a minor role in hemostasis. Two endogenous ligands of CLEC-2 have been identified, the transmembrane protein podoplanin and iron-containing porphyrin hemin, which is formed following hemolysis from red blood cells. Other exogenous ligands such as rhodocytin have contributed to our understanding of the role of CLEC-2. Objectives To identify novel CLEC-2 small-molecule ligands to aid therapeutic targeting of CLEC-2. Methods ALPHA screen technology has been used for the development of a high-throughput screening (HTS) assay recapitulating the podoplanin–CLEC-2 interaction. Light transmission aggregometry was used to evaluate platelet aggregation. Immunoprecipitation and western blot were used to evaluate direct phosphorylation of CLEC-2 and downstream protein phosphorylation. Autodock vina software was used to predict the molecular binding site of katacine and mass spectrometry to determine the polymeric nature of the ligand. Results and Conclusion We developed a CLEC-2–podoplanin interaction assay in a HTS format and screened 5,016 compounds from a European Union-open screen library. We identified katacine, a mixture of polymers of proanthocyanidins, as a novel ligand for CLEC-2 and showed that it induces platelet aggregation and CLEC-2 phosphorylation via Syk and Src kinases. Platelet aggregation induced by katacine is inhibited by the anti-CLEC-2 monoclonal antibody fragment AYP1 F(ab)′2. Katacine is a novel nonprotein ligand of CLEC-2 that could contribute to a better understanding of CLEC-2 activation in human platelets.

Abstract 2977: Profiling oncogenic Ras mutant drugs with homogeneous bioluminescent immunoassays

Abstract Activating KRAS mutations present in many cancers are difficult to target with drugs until recently with the FDA approval of sotorasib (AMG-510), a KRAS G12C selective inhibitor. In an effort to study Ras inhibitors in cells and identify new compounds that inhibit Ras and its downstream signaling, assays such as western or ELISA are commonly used. These traditional immunoassays can be tedious, require multiple washing steps, and are not easily adaptable to a high throughput screening (HTS) format. To overcome these limitations, we developed Lumit, a novel immunoassay approach that combines bioluminescent enzyme subunit complementation technology and immunodetection. We applied this bioluminescent and homogeneous “Add and Read” assay to analyze Ras signaling pathway activation and inhibition through multiple nodes of the pathway including detection of phosphorylated ERK. The assay was used to profile different allele specific inhibitors with cell lines harboring different activating KRAS mutations and obtained the predicted pharmacology and selectivity. The potency of PROTAC compound targeting selective degradation of KRAS G12C was also tested using Lumit and the result showed that maximal decrease in RAS signaling was achieved. Unlike current assays that require lengthy sample preparation and multiple washing steps, the Lumit immunoassay provides an alternative platform because it is homogeneous and allows to decipher signaling pathways activities in a fast and simple manner. Our results demonstrate that this bioluminescent technology can be adapted to any signaling pathway node, allowing scientists to streamline the analysis of signaling pathways of interest such as Ras, and identify much needed inhibitors of its mutants. Citation Format: Hicham Zegzouti, Laurie Engel, Matthew Swiatnicki, Juliano Alves, Said A. Goueli. Profiling oncogenic Ras mutant drugs with homogeneous bioluminescent immunoassays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2977.

Profiling oncogenic KRAS mutant drugs with a cell-based Lumit p-ERK immunoassay.

KRAS is one of the most heavily mutated oncogenes in cancer and targeting mutant KRAS with drugs has proven difficult. However, recent FDA approval of the KRAS G12C selective inhibitor sotorasib (AMG-510), has breathed new life into the drive to develop mutant KRAS inhibitors. In an effort to study RAS inhibitors in cells and identify new compounds that inhibit Ras signaling, western blotting and ELISA assays are commonly used. These traditional immunoassays are tedious, require multiple washing steps, and are not easily adaptable to a high throughput screening (HTS) format. To overcome these limitations, we applied Lumit immunoassay technology to analyze RAS signaling pathway activation and inhibition through the detection of phosphorylated ERK. The assay we developed was used to rank order potencies of allele specific inhibitors within cell lines harboring various activating KRAS mutations. An inhibition profile was obtained indicating various potencies and selectivity of the inhibitors, including MRTX-1133, which was shown to be highly potent against KRAS G12D signaling. MRTX-1133 had approximately 40 and 400 times less inhibitory potency against G12C and G12V mutant KRAS, respectively, while no inhibition of WT KRAS was observed. The potency of PROTAC compound LC-2 targeting selective degradation of KRAS G12C was also tested using the Lumit pERK immunoassay, and a maximal decrease in RAS signaling was achieved. Lumit immunoassays provide a rapid, homogeneous platform for detecting signaling pathway activation and inhibition. Our results demonstrate that this bioluminescent technology can streamline the analysis of signaling pathways of interest, such as RAS-dependent pathways, and be used to identify much needed inhibitors. The results further imply that similar assay designs could be applied to other signaling pathway nodes.

Profiling Oncogenic Ras Mutant Drugs with Homogeneous Bioluminescent Immunoassays

Activating KRAS mutations present in many cancers are difficult to target with drugs until recently with the FDA approval of sotorasib (AMG‐510), a KRAS G12C selective inhibitor. In an effort to study Ras inhibitors in cells and identify new compounds that inhibit Ras and its downstream signaling, assays such as western or ELISA are commonly used. These traditional immunoassays can be tedious, require multiple washing steps, and are not easily adaptable to a high throughput screening (HTS) format. To overcome these limitations, we developed Lumit, a novel immunoassay approach that combines bioluminescent enzyme subunit complementation technology and immunodetection. We applied this bioluminescent and homogeneous “Add and Read” assay to analyze Ras signaling pathway activation and inhibition through multiple nodes of the pathway including detection of phosphorylated ERK. The assay was used to profile different allele specific inhibitors with cell lines harboring different activating KRAS mutations and obtained the predicted pharmacology and selectivity. The potency of PROTAC compound targeting selective degradation of KRAS G12C was also tested using Lumit and the result showed that maximal decrease in RAS signaling was achieved. Unlike current assays that require lengthy sample preparation and multiple washing steps, the Lumit immunoassay provides an alternative platform because it is homogeneous and allows to decipher signaling pathways activities in a fast and simple manner. Our results demonstrate that this bioluminescent technology can be adapted to any signaling pathway node, allowing scientists to streamline the analysis of signaling pathways of interest such as Ras, and identify much needed inhibitors of its mutants.

Long noncoding RNAs in induced pluripotent stem cells and their differentiation.

The breakthrough technology for reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has created a new path for science and medicine. The iPSC technology provides a powerful tool for elucidating the mechanisms of cellular differentiation and cell fate decision as well as to study targets and pathways relevant to pathological processes. As they can be generated from any person, iPSCs are a promising resource for regenerative medicine potentiating the possibility to discover new drugs in a high-throughput screening format and treat diseases through personalized cell therapy-based strategies. However, the reprogramming process is complex, and its regulation needs fine tuning. The regulatory mechanisms of cell reprogramming and differentiation are still not elucidated, but significant results show that multiple long noncoding RNAs (lncRNAs) play essential roles. In this mini-review, we discuss the latest research on lncRNAs in iPSC stemness, neuronal, and cardiac differentiation.

Cell-Based Assays to Identify Modulators of Nrf2/ARE Pathway.

The nuclear factor erythroid 2-related factor (Nrf2) and antioxidant response element (ARE) signaling pathway play an important role in the amelioration of cellular oxidative stress. Thus, assays that detect this pathway can be useful for identifying chemicals that induce or inhibit oxidative stress signaling. This chapter is to describe two cell-based Nrf2/ARE assays in a quantitative high-throughput screening (HTS) format to test a large collection of chemicals for oxidative stress induction ability. The assay descriptions involve cell handling, assay preparation, instrument usage, and assay procedure.

A Quantitative High-Throughput Screening Data Analysis Pipeline for Activity Profiling.

The U.S. Tox21 program has developed in vitro assays to test large collections of environmental chemicals in a quantitative high-throughput screening (qHTS) format, using triplicate 15-dose titrations to generate over 100 million data points to date. Counterscreens are also employed to minimize interferences from non-target-specific assay artifacts, such as compound autofluorescence and cytotoxicity. New data analysis approaches are needed to integrate these data and characterize the activities observed from these assays. Here, we describe a complete analysis pipeline that evaluates these qHTS data for technical quality in terms of signal reproducibility. We integrate signals from repeated assay runs, primary readouts and counterscreens to produce a final call on on-target compound activity.

Development of a single-step fluorogenic sirtuin assay and its applications for high-throughput screening.

Sirtuins (SIRTs) are a class of nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylases. Since SIRTs have different subcellular locations and different preferences for deacylation activity, SIRTs are not only highly gaining significance in biological functions but also implications in human diseases. Therefore, it is valuable to establish a high-throughput screening method for the rapid and accurate discovery of SIRT modulators. In this study, we designed and synthesized small molecules 4a-d as fluorogenic probes based on the different lysine substrates of SIRTs, which can be recognized and catalyzed by SIRTs and then spontaneous intramolecular transesterification can give the fluorescence. We have undertaken a comprehensive study of these fluorogenic probes with different SIRTs for assay optimization, validation, kinetics, parameters, and applications of high-throughput screening formats. We envision that these probes will provide useful and powerful tools for the highly efficient discovery of more SIRT inhibitors.

Evaluation of a Three-Dimensional Primary Human Hepatocyte Spheroid Model: Adoption and Industrialization for the Enhanced Detection of Drug-Induced Liver Injury.

Drug-induced liver injury is a leading cause of compound attrition during both preclinical and clinical drug development, and early strategies are in place to tackle this recurring problem. Human-relevant in vitro models that are more predictive of hepatotoxicity hazard identification, and that could be employed earlier in the drug discovery process, would improve the quality of drug candidate selection and help reduce attrition. We present an evaluation of four human hepatocyte in vitro models of increasing culture complexity (i.e., two-dimensional (2D) HepG2 monolayers, hepatocyte sandwich cultures, three-dimensional (3D) hepatocyte spheroids, and precision-cut liver slices), using the same tool compounds, viability end points, and culture time points. Having established the improved prediction potential of the 3D hepatocyte spheroid model, we describe implementing this model into an industrial screening setting, where the challenge was matching the complexity of the culture system with the scale and throughput required. Following further qualification and miniaturization into a 384-well, high-throughput screening format, data was generated on 199 compounds. This clearly demonstrated the ability to capture a greater number of severe hepatotoxins versus the current routine 2D HepG2 monolayer assay while continuing to flag no false-positive compounds. The industrialization and miniaturization of the 3D hepatocyte spheroid complex in vitro model demonstrates a significant step toward reducing drug attrition and improving the quality and safety of drugs, while retaining the flexibility for future improvements, and has replaced the routine use of the 2D HepG2 monolayer assay at GlaxoSmithKline.