RESUMEN
Diffuse midline glioma (DMG) H3K27-altered is one of the most malignant childhood cancers. Radiation therapy remains the only effective treatment yet provides a 5-year survival rate of only 1%. Several clinical trials have attempted to enhance radiation antitumor activity using radiosensitizing agents, although none have been successful. Given this, there is a critical need for identifying effective therapeutics to enhance radiation sensitivity for the treatment of DMG. Using high-throughput radiosensitivity screening, we identified bromo- and extraterminal domain (BET) protein inhibitors as potent radiosensitizers in DMG cells. Genetic and pharmacologic inhibition of BET bromodomain activity reduced DMG cell proliferation and enhanced radiation-induced DNA damage by inhibiting DNA repair pathways. RNA-Seq and the CUT&RUN (cleavage under targets and release using nuclease) analysis showed that BET bromodomain inhibitors regulated the expression of DNA repair genes mediated by H3K27 acetylation at enhancers. BET bromodomain inhibitors enhanced DMG radiation response in patient-derived xenografts as well as genetically engineered mouse models. Together, our results highlight BET bromodomain inhibitors as potential radiosensitizer and provide a rationale for developing combination therapy with radiation for the treatment of DMG.
Asunto(s)
Histonas , Tolerancia a Radiación , Humanos , Animales , Ratones , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/genética , Histonas/metabolismo , Histonas/genética , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Reparación del ADN/efectos de los fármacos , Glioma/radioterapia , Glioma/patología , Glioma/genética , Glioma/metabolismo , Glioma/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/radioterapia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Fármacos Sensibilizantes a Radiaciones/farmacología , Factores de Transcripción/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Daño del ADN , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas que Contienen Bromodominio , ProteínasRESUMEN
Major depressive disorder is a critical public health problem with a lifetime prevalence of nearly 17% in the United States. One potential therapeutic target is the interaction between hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and an auxiliary subunit of the channel named tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). HCN channels regulate neuronal excitability in the mammalian hippocampus, and recent work has established that antagonizing HCN function rescues cognitive impairment caused by chronic stress. Here, we utilize a high-throughput virtual screen to find small molecules capable of disrupting the TRIP8b-HCN interaction. We found that the hit compound NUCC-0200590 disrupts the TRIP8b-HCN interaction in vitro and in vivo. These results provide a compelling strategy for developing new small molecules capable of disrupting the TRIP8b-HCN interaction.
Asunto(s)
Trastorno Depresivo Mayor , Animales , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Trastorno Depresivo Mayor/metabolismo , Hipocampo/metabolismo , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Mamíferos/metabolismo , Neuronas/metabolismoRESUMEN
Triple-negative breast cancer (TNBC) is the breast cancer subtype with the poorest clinical outcome. The PIM family of kinases has emerged as a factor that is both overexpressed in TNBC and associated with poor outcomes. Preclinical data suggest that TNBC with an elevated MYC expression is sensitive to PIM inhibition. However, clinical observations indicate that the efficacy of PIM inhibitors as single agents may be limited, suggesting the need for combination therapies. Our screening effort identifies PIM and the 20S proteasome inhibition as the most synergistic combination. PIM inhibitors, when combined with proteasome inhibitors, induce significant antitumor effects, including abnormal accumulation of poly-ubiquitinated proteins, increased proteotoxic stress, and the inability of NRF1 to counter loss in proteasome activity. Thus, the identified combination could represent a rational combination therapy against MYC-overexpressing TNBC that is readily translatable to clinical investigations.
Asunto(s)
Neoplasias de la Mama Triple Negativas , Línea Celular Tumoral , Proliferación Celular , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-pim-1 , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Chemical-genetic interaction profiling in model organisms has proven powerful in providing insights into compound mechanism of action and gene function. However, identifying chemical-genetic interactions in mammalian systems has been limited to low-throughput or computational methods. Here, we develop Quantitative and Multiplexed Analysis of Phenotype by Sequencing (QMAP-Seq), which leverages next-generation sequencing for pooled high-throughput chemical-genetic profiling. We apply QMAP-Seq to investigate how cellular stress response factors affect therapeutic response in cancer. Using minimal automation, we treat pools of 60 cell types-comprising 12 genetic perturbations in five cell lines-with 1440 compound-dose combinations, generating 86,400 chemical-genetic measurements. QMAP-Seq produces precise and accurate quantitative measures of acute drug response comparable to gold standard assays, but with increased throughput at lower cost. Moreover, QMAP-Seq reveals clinically actionable drug vulnerabilities and functional relationships involving these stress response factors, many of which are activated in cancer. Thus, QMAP-Seq provides a broadly accessible and scalable strategy for chemical-genetic profiling in mammalian cells.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Fenotipo , Animales , Neoplasias de la Mama/genética , Ingeniería Celular , Línea Celular Tumoral , Supervivencia Celular , Ensayos de Selección de Medicamentos Antitumorales , Redes Reguladoras de Genes , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Neoplasias/genética , Biología de Sistemas/métodosRESUMEN
MEK4, mitogen-activated protein kinase kinase 4, is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. With advances in both computer and biological high-throughput screening, selective chemical entities can be discovered. Structure-based quantitative structure-activity relationship (QSAR) modeling often fails to generate accurate models due to poor alignment of training sets containing highly diverse compounds. Here we describe a highly predictive, nonalignment based robust QSAR model based on a data set of strikingly diverse MEK4 inhibitors. We computed the electrostatic potential (ESP) charges using a density functional theory (DFT) formalism of the donor and acceptor atoms of the ligands and hinge residues. Novel descriptors were then generated from the perturbation of the charge densities of the donor and acceptor atoms and were used to model a diverse set of 84 compounds, from which we built a robust predictive model.
Asunto(s)
MAP Quinasa Quinasa 4/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Humanos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad Cuantitativa , Bibliotecas de Moléculas PequeñasRESUMEN
Mnk kinases (Mnk1 and 2) are downstream effectors of Map kinase pathways and regulate phosphorylation of eukaryotic initiation factor 4E. Engagement of the Mnk pathway is critical in acute myeloid leukemia (AML) leukemogenesis and Mnk inhibitors have potent antileukemic properties in vitro and in vivo, suggesting that targeting Mnk kinases may provide a novel approach for treating AML. Here, we report the development and application of a mutation-based induced-fit in silico screen to identify novel Mnk inhibitors. The Mnk1 structure was modeled by temporarily mutating an amino acid that obstructs the ATP-binding site in the Mnk1 crystal structure while carrying out docking simulations of known inhibitors. The hit compounds display activity in Mnk biochemical and cellular assays, including acute myeloid leukemia progenitors. This approach will enable further rational structure-based drug design of new Mnk inhibitors and potentially novel ways of therapeutically targeting this kinase.
Asunto(s)
Descubrimiento de Drogas , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/enzimología , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Herein we report the discovery of a novel series of highly potent and selective mitogen-activated protein kinase kinaseâ 4 (MEK4) inhibitors. MEK4 is an upstream kinase in MAPK signaling pathways that phosphorylates p38 MAPK and JNK in response to mitogenic and cellular stress queues. MEK4 is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. Optimization of this series via structure-activity relationships and molecular modeling led to the identification of compound 6 ff (4-(6-fluoro-2H-indazol-3-yl)benzoic acid), a highly potent and selective MEK4 inhibitor. This series of inhibitors is the first of its kind in both activity and selectivity and will be useful in further defining the role of MEK4 in prostate and other cancers.
Asunto(s)
Indazoles/síntesis química , Indazoles/farmacología , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Evaluación Preclínica de Medicamentos , Humanos , Especificidad por SustratoRESUMEN
MEK4 is an upstream kinase in MAPK signaling pathways where it phosphorylates p38 MAPK and JNK in response to mitogenic and cellular stress queues. MEK4 is overexpressed and induces metastasis in advanced prostate cancer lesions. However, the value of MEK4 as an oncology target has not been pharmacologically validated because selective chemical probes targeting MEK4 have not been developed. Despite a high level of sequence homology in the ATP-binding site, most reported MEK inhibitors are selective for MEK1/2 and display reduced potency toward other MEKs. Here, we present the first functional and binding selectivity-profiling platform of the MEK family. We applied the platform to profile a set of known kinase inhibitors and used the results to develop an in silico approach for small molecule docking against MEK proteins. The docking studies identified molecular features of the ligands and corresponding amino acids in MEK proteins responsible for high affinity binding versus those driving selectivity. WaterLOGSY and saturation transfer difference (STD) NMR spectroscopy techniques were utilized to understand the binding modes of active compounds. Further minor synthetic manipulations provide a proof of concept by showing how information gained through this platform can be utilized to perturb selectivity across the MEK family. This inhibitor-based approach pinpoints key features governing MEK family selectivity and clarifies empirical selectivity profiles for a set of kinase inhibitors. Going forward, the platform provides a rationale for facilitating the development of MEK-selective inhibitors, particularly MEK4 selective inhibitors, and repurposing of kinase inhibitors for probing the structural selectivity of isoforms.
Asunto(s)
Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Sitios de Unión , Simulación por Computador , Ligandos , MAP Quinasa Quinasa 4/antagonistas & inhibidores , Simulación del Acoplamiento Molecular , Unión ProteicaRESUMEN
Mass cytometry is a recently introduced technology that utilizes transition element isotope-tagged antibodies for protein detection on a single-cell basis. By circumventing the limitations of emission spectral overlap associated with fluorochromes utilized in traditional flow cytometry, mass cytometry currently allows measurement of up to 40 parameters per cell. Recently, a comprehensive mass cytometry analysis was described for the hematopoietic differentiation program in human bone marrow from a healthy donor. The current study describes approaches to delineate cell cycle stages utilizing 5-iodo-2-deoxyuridine (IdU) to mark cells in S phase, simultaneously with antibodies against cyclin B1, cyclin A, and phosphorylated histone H3 (S28) that characterize the other cell cycle phases. Protocols were developed in which an antibody against phosphorylated retinoblastoma protein (Rb) at serines 807 and 811 was used to separate cells in G0 and G1 phases of the cell cycle. This mass cytometry method yielded cell cycle distributions of both normal and cancer cell populations that were equivalent to those obtained by traditional fluorescence cytometry techniques. We applied this to map the cell cycle phases of cells spanning the hematopoietic hierarchy in healthy human bone marrow as a prelude to later studies with cancers and other disorders of this lineage.
Asunto(s)
Puntos de Control del Ciclo Celular , Análisis de la Célula Individual/métodos , Animales , Anticuerpos/química , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/fisiología , Diferenciación Celular , Línea Celular , Proliferación Celular , Separación Celular , Ciclina A/metabolismo , Ciclina B1/metabolismo , Replicación del ADN , Citometría de Flujo , Hematopoyesis , Histonas/metabolismo , Humanos , Inmunofenotipificación , Proteínas de la Membrana/metabolismo , Ratones , Proteína de Retinoblastoma/metabolismo , Coloración y Etiquetado , Linfocitos T/metabolismo , Linfocitos T/fisiología , Elementos de Transición/químicaRESUMEN
Hematopoietic stem cells (HSCs) must exhibit tight regulation of both self-renewal and differentiation to maintain homeostasis of the hematopoietic system as well as to avoid aberrations in growth that may result in leukemias or other disorders. In this study, we sought to understand the molecular basis of lineage determination, with particular focus on factors that influence megakaryocyte/erythrocyte-lineage commitment, in hematopoietic stem and progenitor cells. We used intracellular flow cytometry to identify two novel hematopoietic progenitor populations within the mouse bone-marrow cKit(+) Lineage (-) Sca1(+) (KLS) Flk2 (+) compartment that differ in their protein-level expression of GATA1, a critical megakaryocyte/erythrocyte-promoting transcription factor. GATA1-high repopulating cells exhibited the cell surface phenotype KLS Flk2(+ to int), CD150(int), CD105(+), cMPL(+), and were termed "FSE cells." GATA1-low progenitors were identified as KLS Flk2(+), CD150(-), and cMPL(-), and were termed "Flk(+) CD150(-) cells." FSE cells had increased megakaryocyte/platelet potential in culture and transplant settings and exhibited a higher clonal frequency of colony-forming unit-spleen activity compared with Flk(+) CD150(-) cells, suggesting functional consequences of GATA1 upregulation in promoting megakaryocyte and erythroid lineage priming. Activation of ERK and AKT signal-transduction cascades was observed by intracellular flow cytometry in long-term HSCs and FSE cells, but not in Flk(+) CD150(-) cells in response to stimulation with thrombopoietin, an important megakaryocyte-promoting cytokine. We provide a mechanistic rationale for megakaryocyte/erythroid bias within KLS Flk2(+) cells, and demonstrate how assessment of intracellular factors and signaling events can be used to refine our understanding of lineage commitment during early definitive hematopoiesis.
Asunto(s)
Factor de Transcripción GATA1/metabolismo , Células Madre Hematopoyéticas/metabolismo , Receptores de Trombopoyetina/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Citometría de Flujo , Factor de Transcripción GATA1/genética , Células Madre Hematopoyéticas/efectos de los fármacos , Interleucina-3/farmacología , Ratones , Receptores de Trombopoyetina/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor de Células Madre/farmacología , Trombopoyetina/farmacologíaRESUMEN
Fluorescent cell barcoding (FCB) enables high throughput, high content flow cytometry by multiplexing samples prior to staining and acquisition on the cytometer. Individual cell samples are barcoded, or labeled, with unique signatures of fluorescent dyes so that they can be mixed together, stained, and analyzed as a single sample. By mixing samples prior to staining, antibody consumption is typically reduced 10- to 100-fold. In addition, data robustness is increased through the combination of control and treated samples, which minimizes pipetting error, staining variation, and the need for normalization. Finally, speed of acquisition is enhanced, enabling large profiling experiments to be run with standard cytometer hardware. In this unit, we outline the steps necessary to apply the FCB method to cell lines, as well as primary peripheral blood samples. Important technical considerations, such as choice of barcoding dyes, concentrations, labeling buffers, compensation, and software analysis, are discussed.
Asunto(s)
Células/citología , Citometría de Flujo/métodos , Animales , Separación Celular/métodos , Células Cultivadas , Fluorescencia , Colorantes Fluorescentes/farmacología , Humanos , Modelos Biológicos , Coloración y Etiquetado/métodosRESUMEN
Intracellular flow cytometry permits quantitation of diverse molecular targets at the single-cell level. However, limitations in detection sensitivity inherently restrict the method, sometimes resulting in the inability to measure proteins of very low abundance or to differentiate cells expressing subtly different protein concentrations. To improve these measurements, an enzymatic amplification approach called tyramide signal amplification (TSA) was optimized for assessment of intracellular kinase cascades. First, Pacific Blue, Pacific Orange, and Alexa Fluor 488 tyramide reporters were shown to exhibit low nonspecific binding in permeabilized cells. Next, the effects of antibody concentration, tyramide concentration, and reaction time on assay resolution were characterized. Use of optimized TSA resulted in a 10-fold or greater improvement in measurement resolution of endogenous Erk and Stat cell signaling pathways relative to standard, nonamplified detection. TSA also enhanced assay sensitivity and, in conjunction with fluorescent cell barcoding, improved assay performance according to a metric used to evaluate high-throughput drug screens. TSA was used to profile Stat1 phosphorylation in primary immune system cells, which revealed heterogeneity in various populations, including CD4+ FoxP3+ regulatory T cells. We anticipate the approach will be broadly applicable to intracellular flow cytometry assays with low signal-to-noise ratios.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Citometría de Flujo/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Tiramina/química , Animales , Línea Celular Tumoral , Separación Celular , Quinasas MAP Reguladas por Señal Extracelular/análisis , Femenino , Humanos , Interferón gamma/farmacología , Interleucina-6/farmacología , Células Jurkat , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Límite de Detección , Linfoma/tratamiento farmacológico , Linfoma/metabolismo , Ratones , Ratones Endogámicos BALB C , Fosfoproteínas , Fosforilación , Unión Proteica , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Coloración y Etiquetado/métodos , Tiramina/análisis , Células U937RESUMEN
Drug screening is often limited to cell-free assays involving purified enzymes, but it is arguably best applied against systems that represent disease states or complex physiological cellular networks. Here, we describe a high-content, cell-based drug discovery platform based on phosphospecific flow cytometry, or phosphoflow, that enabled screening for inhibitors against multiple endogenous kinase signaling pathways in heterogeneous primary cell populations at the single-cell level. From a library of small-molecule natural products, we identified pathway-selective inhibitors of Jak-Stat and MAP kinase signaling. Dose-response experiments in primary cells confirmed pathway selectivity, but importantly also revealed differential inhibition of cell types and new druggability trends across multiple compounds. Lead compound selectivity was confirmed in vivo in mice. Phosphoflow therefore provides a unique platform that can be applied throughout the drug discovery process, from early compound screening to in vivo testing and clinical monitoring of drug efficacy.
Asunto(s)
Citometría de Flujo/métodos , Fósforo/análisis , Animales , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Humanos , Quinasas Janus/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factores de Transcripción STAT/metabolismo , Sensibilidad y Especificidad , Transducción de SeñalRESUMEN
Recently, phosphospecific flow cytometry has emerged as a powerful tool to analyze intracellular signaling events in complex populations of cells because of its ability to simultaneously discriminate cell types based on surface marker expression and measure levels of intracellular phosphoproteins. This has provided novel insights into the cell- and pathway-specific nature of immune signaling. However, we and others have found that the fixation and permeabilization steps necessary for phosphoprotein analysis often negatively affect the resolution of cell types based on surface marker analysis and light scatter characteristics. Therefore, we performed a comprehensive profile of >35 different murine surface marker Abs to understand the effects of fixation and permeabilization on surface Ag staining. Fortuitously, approximately 80% of the Abs tested resolved cell populations of interest, although with decreased separation between positive and negative populations and at very different titers than those used on live cells. The other 20% showed either complete loss of separation between populations or loss of intermediately staining populations. We were able to rescue staining of several of these Ags by performing staining after fixation, but before permeabilization, although with limited fluorophore choices. Scatter characteristics of lymphocytes were well retained, but changed dramatically for monocyte and neutrophil populations. These results compile a comprehensive resource for researchers interested in applying phosphospecific flow cytometry to complex populations of cells while outlining steps necessary to successfully apply new surface marker Abs to this platform.
Asunto(s)
Antígenos de Superficie/análisis , Citometría de Flujo , Líquido Intracelular/química , Líquido Intracelular/inmunología , Fosfoproteínas/análisis , Animales , Antígenos de Superficie/inmunología , Antígenos de Superficie/metabolismo , Sitios de Unión de Anticuerpos , Biomarcadores/análisis , Biomarcadores/metabolismo , Permeabilidad de la Membrana Celular/inmunología , Células Cultivadas , Fijadores , Citometría de Flujo/métodos , Colorantes Fluorescentes/análisis , Formaldehído/química , Masculino , Metanol/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Fosforilación , Coloración y EtiquetadoRESUMEN
The third complementarity-determining regions (CDR3s) of antibodies and T cell receptors (TCRs) have been shown to play a major role in antigen binding and specificity. Consistent with this notion, we demonstrated previously that high-affinity, peptide-specific TCRs could be generated in vitro by mutations in the CDR3alpha region of the 2C TCR. In contrast, it has been argued that CDR1 and CDR2 are involved to a greater extent than CDR3s in the process of MHC restriction, due to their engagement of MHC helices. Based on this premise, we initiated the present study to explore whether higher affinity TCRs generated through mutations in these CDRs or other regions would lead to significant reductions in peptide specificity (i.e. the result of greater binding energy gained through interactions with major histocompatibility complex (MHC) helices). Yeast-display technology and flow sorting were used to select high-affinity TCRs from libraries of CDR mutants or random mutants. High-affinity TCRs with mutations in the first residue of the Valpha, CDR1, CDR2, or CDR3 were isolated. Unexpectedly, every TCR mutant, including those in CDR1 and CDR2, retained remarkable peptide specificity. Molecular modeling of various mutants suggested that such exquisite specificity may be due to: (1) enhanced electrostatic interactions with key peptide or MHC residues; or (2) stabilization of CDRs in specific conformations. The results indicate that the TCR is positioned so that virtually every CDR can contribute to the antigen-specificity of a T cell. The conserved diagonal docking of TCRs could thus orient each CDR loop to sense the peptide directly or indirectly through peptide-induced effects on the MHC.
Asunto(s)
Regiones Determinantes de Complementariedad/genética , Regiones Determinantes de Complementariedad/inmunología , Mutación/genética , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Secuencia de Aminoácidos , Regiones Determinantes de Complementariedad/química , Biblioteca de Genes , Modelos Moleculares , Estructura Terciaria de Proteína , Receptores de Antígenos de Linfocitos T/genética , Saccharomyces cerevisiae/genética , Sensibilidad y Especificidad , Especificidad por SustratoRESUMEN
Here we present phospho-specific flow cytometry as a new tool for drug discovery with applications throughout the drug development pipeline, from target identification to library screening, disease model assessment and clinical screening and diagnostics. The single cell, multiparameter nature of flow cytometry generates high-content datasets, and current improvements in the technology are rapidly increasing its high-throughput capacity, making it a valuable platform in modern drug discovery.: