Robust Cell-based Assay Research Articles

Abstract 2065: Development of a genetically validated, cell-based reporter assay for ADAR1 editing activity

Abstract Adenosine Deaminase Acting on RNA (ADAR) enzymes perform adenosine to inosine base editing in RNA, particularly targeting adenosines located within a specific double-stranded stem-loop motif. The ADAR1 gene encodes both a universally expressed isoform (p110) and an interferon-inducible isoform (p150), which plays a role in innate immunity by mediating interferon signaling. In the context of healthy, uninfected cells, ADAR1 performs A-to-I editing on endogenous double-stranded RNA to prevent it from activating downstream dsRNA sensors RIG-I and MDA5, which in-turn activate a pro-inflammatory response. Loss of function mutations in ADAR1 result in aberrant activation of the dsRNA sensors and are involved in autoimmune disorders. ADAR1 dysfunction also impacts cancer cell growth, proliferation, and response to immunotherapy. ADAR1 expression is increased in several types of cancer and ADAR1 knock-out has been demonstrated to improve the response to certain immunotherapies such PD-1/PD-L1 blockade and to circumvent tumor immunotherapy resistance mechanisms, making ADAR1 an attractive target for therapeutic development. We have developed and validated a high-throughput, cell-based assay for measuring ADAR1 editing activity. Several ADAR editing reporter constructs were designed and evaluated for their relative response to ADAR1 over-expression. They all feature an ADAR1 hairpin target with a stop codon (UAG) which is susceptible to ADAR1-mediated editing to a tryptophan (UUG), located upstream of a firefly luciferase gene. We show that increasing transient expression of ADAR1 led to proportional increased activity of the downstream firefly luciferase, indicating dose-dependent transcript editing by ADAR1. Selected reporters were used in conjunction with ADAR2 overexpression to identify an ADAR1-biased reporter. Thus, a reporter with low luciferase activity in response to ADAR2 expression was chosen to establish stable HEK293 cell lines (which express low levels of ADAR1) with and without constitutively expressed ADAR1 protein. ADAR1-overexpressing HEK293 cells displayed high luciferase activity, in accordance with high ADAR1-mediated editing of the reporter transcript, which was decreased in response to siRNA-mediated knockdown of ADAR1 expression or pharmacological inhibition of ADAR1 activity. Sequencing of the reporter RNA confirmed that changes in reporter activity were correlated with editing of the RNA transcript. Finally, these validated ADAR1 editing reporter cell lines were further engineered to constitutively express Renilla luciferase under the control of a CMV promoter, serving as an internal control for the toxicity of ADAR1 inhibitors. In conclusion, our robust cell-based assay provides a well-validated resource to identify modulators of ADAR1 editing activity in a cellular context. Citation Format: Valerie Sapp, Jim Bilakovics, Ava Burr, Fernando Martins, Pavel Shashkin, Veronique Baron, Henry Zhu, Junguk Park. Development of a genetically validated, cell-based reporter assay for ADAR1 editing activity [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 2065.

Abstract 6049: Cell-based PROTAC screening for cancer drug discovery

Abstract Proteolysis-Targeting Chimeras (PROTACs) have emerged as a promising strategy for selectively degrading specific target proteins, thereby offering a novel approach to precision cancer therapy. Among the most notable oncogenes, KRAS with the G12C mutation is responsible for 45%-50% of KRAS mutations in non-small cell lung carcinomas. Additionally, BRD4, a well-studied member of the Bromo- and Extra-Terminal (BET) protein family, is implicated in various hematological and solid tumors. The implementation of cell-based screening for PROTACs targeting either KRAS(G12C) or BRD4 represents a highly valuable platform for advancing cancer drug discovery. We utilized the NanoBRETTM Ternary Complex Assay, NanoBiTTM Assay, and Western Blot assay to evaluate the efficacy of PROTACs. The NanoBRETTM Ternary Complex Assay revealed that KRAS G12C degrader-1 and LC-2 effectively degrade KRAS(G12C), while dBET6 and MZ-1 demonstrated the ability to degrade BRD4. Notably, the NanoBiTTM assay showed that LC-2 dose-dependently degrades KRAS(G12C) in HEK293 cells co-transfected with KRAS(G12C)-LgBiT and KRAS(G12C)-SmBiT. Moreover, BET6 exhibited a dose-dependent degradation of endogenous BRD4 in HepG2 cells, and LC-2 demonstrated the degradation of endogenous KRAS(G12C) in MiaPaCa2 cells. In summary, the development of a robust cell-based assay platform for high-throughput screening of PROTACs marks a substantial advancement in cancer drug discovery. This approach not only enables the identification of promising PROTACs but also holds the potential for more effective and targeted interventions in the ongoing battle against cancer. Citation Format: Yong Wan, Haiqing Ma, Jianghong Wu. Cell-based PROTAC screening for cancer drug discovery [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 6049.

Development of a robust cell-based potency assay for a coxsackievirus A21 oncolytic virotherapy

Oncolytic viruses (OV) are part of a burgeoning field of investigational oncolytic therapy (OT), in which lytic viruses dissolve advanced tumors productively and specifically. One such OT is a Coxsackievirus A21 (CVA21) based OV that is currently under clinical evaluation. A tissue culture infectious dose (TCID50) assay was used for CVA21 potency release and stability testing in early clinical development. The titer measured in this method was an extrapolated value from cytopathic effect (CPE) observed during the serial dilution but doesn't represent direct viral killing of cells. Moreover, the assay was not deemed to be optimal to carry into late phase clinical development due to limitations in assay precision, turn-around time, and sample throughput. To address these points, we developed a plaque assay to measure viral plaque forming units to measure the potency value for drug substance (DS), drug product (DP) and virus seed (master and working) stocks. In this manuscript, we describe the steps taken to develop this plaque assay for the late-stage clinical development, which include the assay qualification, validation, and robustness protocols, and describe statistical methods for data analysis. Moreover, the method was validated for linearity, accuracy, precision, and specificity. Furthermore, the plaque assay quantifies OV infectivity with better precision (32% vs 58%), with higher sample throughput (22 samples/week vs 3 samples/week) and shorter assay turnaround time (4 days vs 7 days) than the TCID50 method. This assay development strategy can provide guidance for the development of robust cell-based potency methods for OVs and other infectious viral products.

TTLL12 has a potential oncogenic activity, suppression of ligation of nitrotyrosine to the C-terminus of detyrosinated α-tubulin, that can be overcome by molecules identified by screening a compound library.

Tubulin tyrosine ligase 12 (TTLL12) is a promising target for therapeutic intervention since it has been implicated in tumour progression, the innate immune response to viral infection, ciliogenesis and abnormal cell division. It is the most mysterious of a fourteen-member TTL/TTLL family, since, although it is the topmost conserved in evolution, it does not have predicted enzymatic activities. TTLL12 seems to act as a pseudo-enzyme that modulates various processes indirectly. Given the need to target its functions, we initially set out to identify a property of TTLL12 that could be used to develop a reliable high-throughput screening assay. We discovered that TTLL12 suppresses the cell toxicity of nitrotyrosine (3-nitrotyrosine) and its ligation to the C-terminus of detyrosinated α-tubulin (abbreviated to ligated-nitrotyrosine). Nitrotyrosine is produced by oxidative stress and is associated with cancer progression. Ligation of nitrotyrosine has been postulated to be a check-point induced by excessive cell stress. We found that the cytotoxicities of nitrotyrosine and tubulin poisons are independent of one another, suggesting that drugs that increase nitrotyrosination could be complementary to current tubulin-directed therapeutics. TTLL12 suppression of nitrotyrosination of α-tubulin was used to develop a robust cell-based ELISA assay that detects increased nitrotyrosination in cells that overexpress TTLL12 We adapted it to a high throughput format and used it to screen a 10,000 molecule World Biological Diversity SETTM collection of low-molecular weight molecules. Two molecules were identified that robustly activate nitrotyrosine ligation at 1 μM concentration. This is the pioneer screen for molecules that modulate nitrotyrosination of α-tubulin. The molecules from the screen will be useful for the study of TTLL12, as well as leads for the development of drugs to treat cancer and other pathologies that involve nitrotyrosination.

Rapid resistance profiling of SARS-CoV-2 protease inhibitors

Resistance to nirmatrelvir (Paxlovid) has been shown by multiple groups and may already exist in clinical SARS-CoV-2 isolates. Here a robust cell-based assay is used to determine the relative potencies of nirmatrelvir, ensitrelvir, and FB2001 against a panel of SARS-CoV-2 main protease (Mpro) variants. The results reveal that these three drugs have at least partly distinct resistance mutation profiles and raise the possibility that the latter compounds may be effective in some instances of Paxlovid resistance and vice versa.

Development and Characterization of an In Vitro Cell-Based Assay to Predict Potency of mRNA-LNP-Based Vaccines.

Messenger RNA (mRNA) vaccines have emerged as a flexible platform for vaccine development. The evolution of lipid nanoparticles as effective delivery vehicles for modified mRNA encoding vaccine antigens was demonstrated by the response to the COVID-19 pandemic. The ability to rapidly develop effective SARS-CoV-2 vaccines from the spike protein genome, and to then manufacture multibillions of doses per year was an extraordinary achievement and a vaccine milestone. Further development and application of this platform for additional pathogens is clearly of interest. This comes with the associated need for new analytical tools that can accurately predict the performance of these mRNA vaccine candidates and tie them to an immune response expected in humans. Described here is the development and characterization of an imaging based in vitro assay able to quantitate transgene protein expression efficiency, with utility to measure lipid nanoparticles (LNP)-encapsulated mRNA vaccine potency, efficacy, and stability. Multiple biologically relevant adherent cell lines were screened to identify a suitable cell substrate capable of providing a wide dose-response curve and dynamic range. Biologically relevant assay attributes were examined and optimized, including cell monolayer morphology, antigen expression kinetics, and assay sensitivity to LNP properties, such as polyethylene glycol-lipid (or PEG-lipid) composition, mRNA mass, and LNP size. Collectively, this study presents a strategy to quickly optimize and develop a robust cell-based potency assay for the development of future mRNA-based vaccines.

Evaluation of (Anti)androgenic Activities of Environmental Xenobiotics in Milk Using a Human Liver Cell Line and Androgen Receptor-Based Promoter-Reporter Assay

The recent reports on milk consumption and its associated risk with hormone related disorders necessitates the evaluation of dairy products for the presence of endocrine disrupting chemicals (EDCs) and ensure the safety of consumers. In view of this, we investigated the possible presence of (anti)androgenic contaminants in raw and commercialized milk samples. For this purpose, a novel HepARE-Luc cell line that stably expresses human androgen receptor (AR) and the androgen responsive luciferase reporter gene was generated and used in the present study. Treatment of this cell line with androgens and corresponding antiandrogen (flutamide) stimulated or inhibited expression of reporter luciferase, respectively. Real time polymerase chain reaction and immunostaining results exhibited transcription response and translocation of AR from the cytoplasm to the nucleus in response to androgen. Observations implied that a cell-based xenobiotic screening assay via AR response can be conducted for assessing the (anti)androgenic ligands present in food chain including milk. Therefore, the cell line was further used to screen the (anti)androgenic activity of a total of 40 milk fat samples procured as raw or commercial milk. Some of the raw and commercial milk fat samples distinctly showed antiandrogenic activities. Subsequently, some commonly used environmental chemicals were also evaluated for their (anti)androgenic activities. Initial observations with molecular docking studies of experimental compounds were performed to assess their interaction with AR ligand binding domain. Furthermore, (anti)androgenic activities of these compounds were confirmed by performing luciferase assay using the HepARE-Luc cell line. None of the test compounds showed androgenic activities rather some of them like Bisphenol A (BPA) and rifamycin showed antiandrogenic activities. In conclusion, our results provide a valuable information about the assessment of (anti)androgenic activities present in milk samples. Overall, it is proposed that a robust cell-based CALUX assay can be used to assess the (anti)androgenic activities present in milk which can be attributed to different environmental chemicals present therein.

Construction of a culture protocol for functional bile canaliculi formation to apply human iPS cell-derived hepatocytes for cholestasis evaluation

Cholestatic toxicity causes the failure of pharmaceutical agents during drug development and, thus, should be identified at an early stage of drug discovery and development. The formation of functional bile canaliculi in human hepatocytes is required for in vitro cholestasis toxicity tests conducted during the early stage of drug development. In this study, we investigated the culture conditions required for the formation of bile canaliculi using human-induced pluripotent stem cell-derived hepatocytes (hiPSC-Heps). When hiPSC-Heps were sandwich-cultured under the condition we established, extended bile canaliculi were formed on the whole well surfaces. Biliary efflux transporters were localized in the formed bile canaliculi structures which had junctional complexes. After the model substrates of the biliary efflux transporters were taken up into cells, their subsequent excretion into the bile canaliculi was observed and was found to be impeded by each inhibitor of the biliary efflux transporter. These findings suggest that bile canaliculi have transporter-specific bile excretion abilities. We will continue to study the application of this culture protocol to cell-based cholestasis assay system. As a result, the culture protocol could lead to a highly predictable, robust cell-based cholestasis assay system because it forms functional bile canaliculi reproducibly and efficiently.

Complex Tumor Spheroids, a Tissue-Mimicking Tumor Model, for Drug Discovery and Precision Medicine

Malignant tumors are complex tissues composed of malignant cells, vascular cells, structural mesenchymal cells including pericytes and carcinoma-associated fibroblasts, infiltrating immune cells, and others, collectively called the tumor stroma. The number of stromal cells in a tumor is often much greater than the number of malignant cells. The physical associations among all these cell types are critical to tumor growth, survival, and response to therapy. Most cell-based screens for cancer drug discovery and precision medicine validation use malignant cells in isolation as monolayers, embedded in a matrix, or as spheroids in suspension. Medium- and high-throughput screening with multiple cell lines requires a scalable, reproducible, robust cell-based assay. Complex spheroids include malignant cells and two normal cell types, human umbilical vein endothelial cells and highly plastic mesenchymal stem cells, which rapidly adapt to the malignant cell microenvironment. The patient-derived pancreatic adenocarcinoma cell line, K24384-001-R, was used to explore complex spheroid structure and response to anticancer agents in a 96-well format. We describe the development of the complex spheroid assay as well as the growth and structure of complex spheroids over time. Subsequently, we demonstrate successful assay miniaturization to a 384-well format and robust performance in a high-throughput screen. Implementation of the complex spheroid assay was further demonstrated with 10 well-established pancreatic cell lines. By incorporating both human stromal and tumor components, complex spheroids might provide an improved model for tumor response in vivo.

Abstract LB123: A high value pharmacological platform dedicated to the real time study of stimulatory immune checkpoint signaling pathways

Abstract Immune checkpoint (ICP) co-receptors play a key role in the fine modulation of the immune response. While inhibitory ICP co-receptors are mainly characterized in cancer by the exhaustion of the immune response, stimulatory ICP co-receptors are clearly involved in an anarchic activation of the immune system leading to autoimmune diseases. But in another hand, this subtype of co-receptor shows a great asset in cancer therapy by increasing anti-tumoral response. All together, these co-receptors show a great interest for the development of innovative therapies in immunology or immuno-oncology. Nowadays, treatments targeting these co-receptors are currently tested in clinical trials or approved for use in man, especially antibodies targeting both inhibitory and stimulatory ICP. Small molecule-based strategy is tested in early drug discovery and in early clinical trials. In order to screen and assess the potency and efficiency of these therapies, robust and reliable cell-based assays are urgently needed. At Domain Therapeutics, using our powerful proprietary BRET technology: bioSens-AllTM, we successfully develop and validate a cell-based assay platform to assess immune checkpoint pharmacology. The activation or blockade of the signaling of checkpoint co-receptors are monitored in real time and in living cells. BRET assays dedicated to stimulatory ICPs: 4-1BB/4-1BB Ligand and OX-40/OX-40 Ligand were designed. BRET assay development is inspired by the natural recruitment and proximity of specific cytoplasmic partners very close to their co-receptors in an activated pathway. Indeed, the first assay design is based on the co-receptor fused with rGFP and its cytoplasmic partner fused with a rLucII. In the second assay design, the co-receptor is no longer fused, but is co-transfected with a membrane anchor fused with the rGFP and co-transfected with the cytoplasmic partner fused with rLucII. So, in an activated system (soluble ligand, antibody or in co-culture system) a modulation of BRET can be observed. For ICP assays dedicated to 4-1BB, BRET signal is triggered using soluble ligand, agonist antibody, or co-culture where TRAF1 is the biosensor fused with rLucII. Further validation was obtained for OX-40/OX-40 Ligand. Those BRET assays complement once previously developed for inhibitory ICPs axis: PD-1/PD-Ligand 1 or PD-Ligand 2 and CTLA-4/CD80-CD86. The ICP Platform set up here shows a good accuracy and robustness and represents a strong and reliable technology for drug discovery dedicated to ICPs. Our spatio-temporal cell-based functional assays can support broad drug programs, including: High Throughput Functional Screening, Lead Optimization and Bioanalytical QC lot Release. Citation Format: Alice GENTIL DIT MAURIN, Christel FRANCHET, Stephan SCHANN, Xavier LEROY. A high value pharmacological platform dedicated to the real time study of stimulatory immune checkpoint signaling pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB123.

Targeting the HuR Oncogenic Role with a New Class of Cytoplasmic Dimerization Inhibitors.

The development of novel therapeutics that exploit alterations in the activation state of key cellular signaling pathways due to mutations in upstream regulators has generated the field of personalized medicine. These first-generation efforts have focused on actionable mutations identified by deep sequencing of large numbers of tumor samples. We propose that a second-generation opportunity exists by exploiting key downstream "nodes of control" that contribute to oncogenesis and are inappropriately activated due to loss of upstream regulation and microenvironmental influences. The RNA-binding protein HuR represents such a node. Because HuR functionality in cancer cells is dependent on HuR dimerization and its nuclear/cytoplasmic shuttling, we developed a new class of molecules targeting HuR protein dimerization. A structure-activity relationship algorithm enabled development of inhibitors of HuR multimer formation that were soluble, had micromolar activity, and penetrated the blood-brain barrier. These inhibitors were evaluated for activity validation and specificity in a robust cell-based assay of HuR dimerization. SRI-42127, a molecule that met these criteria, inhibited HuR multimer formation across primary patient-derived glioblastoma xenolines (PDGx), leading to arrest of proliferation, induction of apoptosis, and inhibition of colony formation. SRI-42127 had favorable attributes with central nervous system penetration and inhibited tumor growth in mouse models. RNA and protein analysis of SRI-42127-treated PDGx xenolines across glioblastoma molecular subtypes confirmed attenuation of targets upregulated by HuR. These results highlight how focusing on key attributes of HuR that contribute to cancer progression, namely cytoplasmic localization and multimerization, has led to the development of a novel, highly effective inhibitor. SIGNIFICANCE: These findings utilize a cell-based mechanism of action assay with a structure-activity relationship compound development pathway to discover inhibitors that target HuR dimerization, a mechanism required for cancer promotion.

Development of an antibody-dependent cellular cytotoxicity reporter assay for measuring anti-Middle East Respiratory Syndrome antibody bioactivity

Middle East Respiratory Syndrome coronavirus (MERS-CoV) is a highly virulent pathogen that causes Middle East Respiratory Syndrome (MERS). Anti-MERS-CoV antibodies play an integral role in the prevention and treatment against MERS-CoV infections. Bioactivity is a key quality attribute of therapeutic antibodies, and high accuracy and precision are required. The major methods for evaluating the antiviral effect of antiviral antibodies include neutralization assays using live viruses or pseudoviruses are highly variable. Recent studies have demonstrated that the antibody-dependent cellular cytotoxicity (ADCC) activity of antiviral antibodies is more consistent with the virus clearance effect in vivo than neutralization activity. However, no reports evaluating the ADCC activity of anti-MERS antibodies have been published to date. Here, we describe the development of a robust and reliable cell-based reporter gene assay for the determination of ADCC activity of anti-MERS antibodies using 293T/MERS cells stably expressing the spike protein of MERS-CoV (MERS-S) as target cells and the engineered Jurkat/NFAT-luc/FcγRIIIa stably expressing FcγRIIIA and NFAT reporter gene as effector cells. According to the ICH-Q2 analytical method guidelines, we carefully optimized the experimental conditions and assessed the performance of our assay. In addition, we found that the ADCC activity of afucosylated anti-MERS antibodies is higher than their fucosylated counterparts. The establishment of this ADCC determination system provides a novel method for evaluating the bioactivity of anti-MERS antibodies and improving ADCC activity through modification of N-glycosylation of the Fc segment.

A simple and robust cell-based assay for the discovery of novel cytokinesis inhibitors.

Cytokinesis is the last step of mitotic cell division that separates the cytoplasm of dividing cells. Small molecule inhibitors targeting either the elements of the regulatory pathways controlling cytokinesis, or the terminal effectors have been of interest as potential drug candidates for the treatment of various diseases. Here we present a detailed protocol for a cell-based cytokinesis assay that can be used for the discovery of novel cytokinesis inhibitors. The assay is performed in a 96-well plate format in 48 h. Living cells, nuclei and nuclei of dead cells are identified by a single staining step using three fluorescent dyes, followed by rapid live cell imaging. The primary signal is the nuclei-to-cell ratio (NCR). In the presence of cytokinesis inhibitors, this ratio increases over time, as the ratio of multinucleated cells increases in the population. The ratio of dead nuclei to total nuclei provides a simultaneous measure of cytotoxicity. A screening window coefficient (Z`) of 0.65 indicates that the assay is suitable for screening purposes, as the positive and negative controls are well-separated. EC50 values can be reliably determined in a single 96-well plate by using only six different compound concentrations, enabling the testing of 4 compounds per plate. An excellent test-retest reliability (R2 = 0.998) was found for EC50 values covering a ~1500-fold range of potencies. Established small molecule inhibitors of cytokinesis operating via direct action on actin dynamics or nonmuscle myosin II are used to demonstrate the robustness, simplicity and flexibility of the assay.

Detailed Analyses of Zika Virus Tropism in Culex quinquefasciatus Reveal Systemic Refractoriness.

The role of Culex quinquefasciatus in Zika virus transmission has been debated since the epidemic of Zika occurred in the Americas in 2015 to 2016. The majority of studies have found no evidence that C. quinquefasciatus or other Culex species are competent vectors of Zika virus, and the few studies that have proposed Zika vector status for C. quinquefasciatus have relied predominantly on quantitative real-time PCR (qRT-PCR) for viral detection. We assessed the infectious range of pre- and post-epidemic Zika virus isolates in order to classify mosquito samples based on titer infectiousness and demonstrated that two strains of C. quinquefasciatus, including one previously found to be competent, are highly resistant to infection with these Zika isolates compared to Aedes aegypti and are not competent for virus transmission. Further dissection of the dynamics of Zika exposure in both A. aegypti and C. quinquefasciatus revealed that while virus transmission by C. quinquefasciatus is blocked at the levels of the midgut and salivary glands, viral RNA persists in these tissues for prolonged periods post-exposure. We assessed Zika entry dynamics in both Aedes and Culex cells, and our results suggest that Zika virus infection in Culex cells may be blocked downstream of cell entry. These findings strongly suggest that C. quinquefasciatus is not a vector of Zika virus and additionally inform the use of qRT-PCR in vector competence assays as well as our understanding of barriers to arbovirus infection in non-susceptible mosquito species.IMPORTANCE Understanding which mosquito species transmit an emerging arbovirus is critical to effective vector control. During the Zika virus epidemic in 2015 to 2016, Aedes mosquitoes were confirmed as vectors. However, studies addressing the vector status of Culex quinquefasciatus mosquitoes presented conflicting evidence and remain an outstanding source of confusion in the field. Here, we established a robust cell-based assay to identify infectious titers of Zika virus and assessed the virus titers in C. quinquefasciatus by quantitative real-time PCR (qRT-PCR). We found that while low levels of virus were detected in C. quinquefasciatus, these titers did not correspond to infectious virus, and these mosquitoes did not transmit virus in the saliva. We also present evidence that the virus may enter Culex cells before infection is disrupted. Our findings are important for future studies incriminating vector species using qRT-PCR for virus detection and offer new information on how virus transmission is blocked by mosquitoes.

Abstract 6308: Development of a pharmacological platform to study in real time immune checkpoints signaling pathways: validation with therapeutic mAbs and small molecules

Abstract Regulation of immune responses is tightly controlled through a balance of co-stimulatory and inhibitory checkpoint receptors, often exploited by many cancers. Therapeutics that block inhibitory receptors or activate immunostimulatory checkpoint receptors have proven to be powerful agents for the restoration of anti-tumor immune responses. While some antibodies targeting these checkpoint receptors have been tested and approved for use in man, many others are in development. For the same targets, there are also small molecules being tested in early clinical trials. In order to screen and assess the potency and efficiency of these therapies, robust and reliable cell-based assays are urgently needed. At Domain Therapeutics, we have successfully developed and validated immune checkpoint pharmacological assays based on our proprietary BRET technology, bioSensAllTM. In these assays, the activation or blockade of the signaling of checkpoint receptors are assessed. PD-1 is expressed on T-cells, while it's ligands, PD-L1 or PD-L2, are expressed on the surface of tumor cells or antigen presenting cells. Like many other immunoglobulin receptors, PD-1 harbors immunoreceptor tyrosine inhibitory motifs (ITIMs) in its cytoplasmic tail that are important signaling motifs. When its ligand, e.g. PD-L1, binds to PD-1, Src family kinases phosphorylate the ITIM motif, resulting in the recruitment of SH2-domain containing phosphatases, SHP-1 and SHP-2, which are involved in inhibiting the T-cell response. As a translation of this concept, we present PD-1 signaling assays using our proprietary BRET technology. For each inhibitory co-receptor, two types of assays were generated. In the first assay, cells expressing the PD-1 and SHP-2 proteins, fused respectively to rGFP and rLucII, are co-incubated with ligand-presenting cells. This results in PD-1 activation and SHP-2 recruitment to the PD-1 receptor, generating a specific BRET signal. In the second assay design, PD-1 positive cells are transfected with SHP-2 and membrane anchor protein, fused respectively with rLucII and rGFP. Upon target engagement following co-culture with cells expressing PD-L1, translocation of SHP-2 close to the membrane triggers a BRET response. Interestingly, the BRET signal is inhibited with either neutralizing antibodies (against PD-L1 or PD-1) or inhibitory small molecules. Further validation was obtained for PD-L2/PD-1 and CD86/CTLA-4. The Immune Checkpoint Platform shows a good accuracy and robustness. These spatio-temporal cell-based functional assays can support broad drug programs, including: High Throughput Functional Screening, Lead Optimization and Bioanalytical QC lot Release. Based on their complementarity, those assays can be used in tandem for primary and secondary screenings and offer a strong and reliable platform for drug discovery. Citation Format: Alice Gentil Dit Maurin, Christel Franchet, Xavier Leroy, Stephane Schann. Development of a pharmacological platform to study in real time immune checkpoints signaling pathways: validation with therapeutic mAbs and small molecules [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6308.

Discovery of Protein-Protein Interaction Inhibitors by Integrating Protein Engineering and Chemical Screening Platforms

Protein-protein interactions (PPIs) govern intracellular life, and identification of PPI inhibitors is challenging. Roadblocks in assay development stemming from weak binding affinities of natural PPIs impede progress in this field. We postulated that enhancing binding affinity of natural PPIs via protein engineering will aid assay development and hit discovery. This proof-of-principle study targets PPI between linear ubiquitin chains and NEMO UBAN domain, which activates NF-κB signaling. Using phage display, we generated ubiquitin variants that bind to the functional UBAN epitope with high affinity, act as competitive inhibitors, and structurally maintain the existing PPI interface. When utilized in assay development, variants enable generation of robust cell-based assays for chemical screening. Top compounds identified using this approach directly bind to UBAN and dampen NF-κB signaling. This study illustrates advantages of integrating protein engineering and chemical screening in hit identification, a development that we anticipate will have wide application in drug discovery.

Abstract 19: Targeting Delta133p53 isoform with small-molecule compounds to modulate cellular senescence

Abstract Cellular senescence (CS) and senescence-associated secretory phenotype (SASP) contribute to aging and age-related diseases. Mechanistically, the stress sensor p53 plays a pivotal role in the initiation and maintenance of CS. In humans, TP53 is expressed as 12 isoforms that contribute to the fine-tuning of p53 activity. Delta133p53 is generated from an alternative promoter located in intron 4 and is therefore devoid of both the transactivation domain and part of the DNA binding domain. Delta133p53 is predominantly located in the nucleus and is largely regulated at the protein level through chaperone-assisted selective autophagic degradation. Delta133p53 counteracts p53-mediated replicative CS and reduces the secretion of SASP cytokines. Delta133p53 is expressed in most normal tissues, but its expression is deregulated in age-associated diseases such as cancer, neurodegenerative diseases, and premature aging disorders. Delta133p53 is downregulated in exhausted CD8+ T cells, nearly senescent fibroblasts from Hutchinson-Gilford progeria syndrome patients, and astrocytes from irradiated brains and Alzheimer’s disease and amyotrophic lateral sclerosis patients. Delta133p53 overexpression extends the replicative lifespan in normal cells but does not cause immortalization or malignant transformation. Hence, delta133p53 appears to be a safe, novel therapeutic target to regulate CS. The present study aims at identifying small-molecule compounds that stabilize or prevent the degradation of delta133p53 protein and inhibit CS. To screen for activators of delta133p53, we developed two cell-based high-throughput screening (HTS) assays using an inducible vector for the expression of an eGFP-tag recombinant protein, and a luminescent peptide tag (HiBiT) appended onto the endogenous gene. The biologic activity of the fusion proteins was confirmed by evaluating the i) nuclear localization, ii) autophagic degradation during CS, and iii) ability to delay replicative CS. In collaboration with the National Center for Advancing Translational Sciences (NCATS), we screened a collection of ~8,350 compounds that includes all drugs that have been approved for use by the US Food and Drug Administration. Selected compounds were then screened in a secondary assay to evaluate their efficacy at reducing SASP cytokines secretion. Several candidate compounds are currently being investigated to confirm their effect on endogenous delta133p53 expression and CS. We have established robust cell-based HTS assays to screen for activators of delta133p53 and identified candidate compounds that could potentially develop into novel therapeutic leads to treat major life-threatening diseases. Citation Format: Delphine Lissa, Kyra Ungerleider, Izumi Horikawa, Patricia Dranchak, Erin Oliphan, Jessica Beck, Sebastien Jo, James Inglese, Curtis C. Harris. Targeting Delta133p53 isoform with small-molecule compounds to modulate cellular senescence [abstract]. In: Proceedings of the AACR Special Conference on Advancing Precision Medicine Drug Development: Incorporation of Real-World Data and Other Novel Strategies; Jan 9-12, 2020; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_1):Abstract nr 19.

Methionyl-tRNA synthetase inhibitor has potent in vivo activity in a novel Giardia lamblia luciferase murine infection model.

Methionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia. To properly analyse the therapeutic potential of the MetRS inhibitor 1717, experimental tools including a robust cell-based assay and a murine model of infection were developed based on novel strains of G. lamblia that employ luciferase reporter systems to quantify viable parasites. Systematic screening of Giardia-specific promoters and luciferase variants led to the development of a strain expressing the click beetle green luciferase. Further modifying this strain to express NanoLuc created a dual reporter strain capable of quantifying parasites in both the trophozoite and cyst stages. These strains were used to develop a high-throughput cell assay and a mouse infection model. A library of MetRS inhibitors was screened in the cell assay and Compound-1717 was tested for efficacy in the mouse infection model. Cell viability in in vitro compound screens was quantified via bioluminescence readouts while infection loads in mice were monitored with non-invasive whole-animal imaging and faecal analysis. Compound-1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which was supported by data from enzymatic and phenotypic cell assays. The new in vitro and in vivo assays based on luciferase expression by engineered G. lamblia strains are useful for the discovery and development of new therapeutics for giardiasis. MetRS inhibitors, as validated by Compound-1717, have promising anti-giardiasis properties that merit further study as alternative therapeutics.

A SMN2 Splicing Modifier Rescues the Disease Phenotypes in an In Vitro Human Spinal Muscular Atrophy Model.

Spinal muscular atrophy (SMA) is caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene. Only ∼10% of the products of SMN2, a paralogue of SMN1, are functional full-length SMN (SMN-FL) proteins, whereas SMN2 primarily produces alternatively spliced transcripts lacking exon 7. Reduced SMN protein levels in SMA patients lead to progressive degeneration of spinal motor neurons (MNs). In this study, we report an advanced platform based on an SMN2 splicing-targeting approach for SMA drug screening and validation using an SMN2 splicing reporter cell line and an in vitro human SMA model through induced pluripotent stem cell (iPSC) technology. Through drug screening using a robust cell-based luciferase assay to quantitatively measure SMN2 splicing, the small-molecule candidate compound rigosertib was identified as an SMN2 splicing modulator that led to enhanced SMN protein expression. The therapeutic potential of the candidate compound was validated in MN progenitors differentiated from SMA patient-derived iPSCs (SMA iPSC-pMNs) as an in vitro human SMA model, which recapitulated the biochemical and molecular phenotypes of SMA, including lower levels of SMN-FL transcripts and protein, enhanced cell death, and reduced neurite length. The candidate compound exerted strong splicing correction activity for SMN2 and potently alleviated the disease-related phenotypes of SMA iPSC-pMNs by modulating various cellular and molecular abnormalities. Our combined screening platform representing a pMN model of human SMA provides an efficient and reliable drug screening system and is a promising resource for drug evaluation and the exploration of drug modes of action.

Cell based functional assays for IDO1 inhibitor screening and characterization.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a new immune-oncology target and its inhibitors have shown promise in the clinic especially in combination with other immune-stimulating agents. Here we describe two robust cell-based assays for screening IDO1 inhibitors. Both assays can be easily adopted by most laboratories and utilized for screening of IDO1 inhibitors. Endogenous IDO1 expression is induced in a cancer cell line with interferon gamma and its activity is assessed by measuring kynurenine secreted into the media. The effect of cancer cell IDO1 induction and inhibition on T cell activation is evaluated in a co-culture assay using Jurkat T cell line. Additional readouts assessing cell viability are employed for early detection of false positive IDO1 inhibitors and toxic compounds. Clinical candidates epacadostat and BMS-986205 were evaluated in the assays as control compounds, the former can completely inhibit IDO1 activity while the maximum effect of the later is limited (to about 80% in our system) consistent with the differences in their interaction with IDO1. Nanomolar concentrations of both compounds rescued IDO1 mediated inhibition of T cell activation. However, treatment with micromolar concentrations of BMS-986205 blocked Jurkat T cell activation and after prolonged incubation induced cell death.