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Enhancing antitumor activities of the human immune system is a clinically proven approach with the advent of monoclonal antibodies recognizing programmed cell death protein-1 (PD1) receptors on immune cell surfaces. Historically, using flow cytometry as a means to assess next-generation agent activities was underused, largely due to limits on cell number and assay sensitivity. Here, we leveraged an IntelliCyt high-throughput flow cytometry platform to monitor human dendritic cell maturation and lymphocyte proliferation in mixed lymphocyte reactions. Specifically, we established flow cytometry-based immunophenotyping and screening methodologies capable of measuring T-cell activation as a result of cell-associated antigens presented on dendritic cell surfaces, as indicated by cell proliferation, cytokine secretion, and surface marker expression. Together, the overall novelty of this 384-well platform is its capability to measure multiple functional readouts in one well and consistently evaluate large numbers of compounds in a single study, as well as its ability to show increased assay sensitivity requiring considerably fewer primary cells and less reagents compared to more traditional 96-well flow cytometry methods.

Chemotaxis is the directional movement of cells in response to a chemical stimulus and is vital for many physiological processes, including immune responses, tumor metastasis, wound healing, and blood vessel formation. Therefore, modulation of chemotaxis is likely to be of therapeutic benefit. Hence, a high-throughput means to conduct chemotaxis assays is advantageous for lead evaluation and optimization in drug discovery. In this study, we have validated a novel approach for a higher-throughput, label-free, image-based IncuCyte chemotaxis assay encompassing various cell types, including T cells, B cells, mouse Th17, immature and mature dendritic cells, monocyte THP-1, CCRF-CEM, monocytes, neutrophils, macrophages, and MDA-MB-231. These assays enable us to visualize chemotactic cell migration in real time and perform kinetic cell motility studies on an automated platform, thereby allowing us to incorporate the quantitative studies of cell migration behavior into a routine drug discovery screening cascade.

Oral agents targeting Janus-associated kinases (JAKs) are promising new agents in clinical development. To better understand the relationship between JAK inhibition and biological outcome, compounds targeting JAKs were evaluated in peripheral human whole blood. To date, these analyses are low throughput and costly. Here, we developed a robust 384-well, high-throughput flow-based assay approach to screen small molecules for JAK/STAT signaling inhibition in human whole blood. This assay platform provides a highly sensitive analysis of signaling events in blood and facilitates measurement of target engagement. Further, the automation technologies and process optimizations developed here overcame sample integrity, handling, and multiparametric data analysis bottlenecks without affecting assay performance. Together these efforts dramatically increased sample throughput compared to conventional manual flow cytometric approaches and enabled development of novel JAK/STAT inhibitors., (© 2016 Society for Laboratory Automation and Screening.)

Cytokines serve as a major mechanism of communication between immune cells and are the functional molecules at the end of immune pathways. Abnormalities in cytokines are involved in a wide variety of diseases, including chronic inflammation, autoimmune diseases, and cancer. Cytokines are not only direct targets of therapeutics but also important biomarkers for assessing drug efficacy and safety. Traditionally, enzyme-linked immunosorbent assays (ELISA) were most popular for identifying and quantifying cytokines. However, ELISA is expensive, labor intensive, and low throughput. Here, we report the development of a miniaturized Luminex (Austin, TX) assay platform to establish a panel of high-throughput, multiplexed assays for measuring cytokines in human whole blood. The miniaturized 384-well Luminex assay uses <25% of the assay reagents compared with the 96-well assay. The development and validation of the 384-well Luminex cytokine assays enabled high-throughput screening of compounds in primary cells using cytokines as physiologically relevant readouts. Furthermore, this miniaturized multiplexed technology platform allows for high-throughput biomarker profiling of biofluids from animal studies and patient samples for translational research., (© 2016 Society for Laboratory Automation and Screening.)

GPCRs remain one of the most important classes of drug targets and, therefore, GPCR assay development and high-throughput GPCR ligand profiling continue to be major efforts in drug discovery. This article focuses on GPCR platform strategies from hits to leads with miniaturized complex pharmacology approaches. Three main areas of GPCR profiling are discussed including pharmacologically relevant hit identification, the pharmacology dossier applied to parallel structure activity and structure liability relationships and high-throughput mechanism studies from genotype to phenotype., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Hetero-oligomeric complexes of G protein-coupled receptors (GPCRs) may represent novel therapeutic targets exhibiting different pharmacology and tissue- or cell-specific site of action compared with receptor monomers or homo-oligomers. An ideal tool for validating this concept pharmacologically would be a hetero-oligomer selective ligand. We set out to develop and execute a 1536-well high-throughput screen of over 1 million compounds to detect potential hetero-oligomer selective ligands using a β-arrestin recruitment assay in U2OS cells coexpressing recombinant µ- and δ-opioid receptors. Hetero-oligomer selective ligands may bind to orthosteric or allosteric sites, and we might anticipate that the formation of hetero-oligomers may provide novel allosteric binding pockets for ligand binding. Therefore, our goal was to execute the screen in such a way as to identify positive allosteric modulators (PAMs) as well as agonists for µ, δ, and hetero-oligomeric receptors. While no hetero-oligomer selective ligands were identified (based on our selection criteria), this single screen did identify numerous µ- and δ-selective agonists and PAMs as well as nonselective agonists and PAMs. To our knowledge, these are the first µ- and δ-opioid receptor PAMs described in the literature., (© 2014 Society for Laboratory Automation and Screening.)

Receptor tyrosine kinases (RTKs) regulate a wide range of important biological activities, including cell proliferation, differentiation, migration, and apoptosis. Abnormalities in RTKs are involved in numerous diseases, including cancer and other proliferative disorders. AXL belongs to the TAM (Tyso3, AXL, and Mer) family of RTKs. The AXL signaling pathway represents an attractive target for the treatment of diseases, such as cancer. Using phospho-AKT as readout, a high-throughput 384-well cell-based assay was established in the NCI-H1299 human non-small cell lung carcinoma cell line to evaluate compound potency in inhibiting AXL pathway activation. In addition, a counter screen assay was established in the same cellular background to differentiate AXL kinase inhibitors from AXL receptor antagonists, which block the interaction of AXL and its natural ligand GAS6. These cell-based functional assays are useful tools in the identification and optimization of small molecules and biological reagents for potential therapeutics for the treatment of GAS6/AXL-related diseases.

Transporter proteins are known to play a critical role in affecting the overall absorption, distribution, metabolism, and excretion characteristics of drug candidates. In addition to efflux transporters (P-gp, BCRP, MRP2, etc.) that limit absorption, there has been a renewed interest in influx transporters at the renal (OATs, OCTs) and hepatic (OATPs, BSEP, NTCP, etc.) organ level that can cause significant clinical drug-drug interactions (DDIs). Several of these transporters are also critical for hepatobiliary disposition of bilirubin and bile acid/salts, and their inhibition is directly implicated in hepatic toxicities. Regulatory agencies took action to address transporter-mediated DDI with the goal of ensuring drug safety in the clinic and on the market. To meet regulatory requirements, advanced bioassay technology and automation solutions were implemented for high-throughput transporter screening to provide structure-activity relationship within lead optimization. To enhance capacity, several functional assay formats were miniaturized to 384-well throughput including novel fluorescence-based uptake and efflux inhibition assays using high-content image analysis as well as cell-based radioactive uptake and vesicle-based efflux inhibition assays. This high-throughput capability enabled a paradigm shift from studying transporter-related issues in the development space to identifying and dialing out these concerns early on in discovery for enhanced mechanism-based efficacy while circumventing DDIs and transporter toxicities.

Tumor cell proliferation assays are widely used for oncology drug discovery, including target validation, lead compound identification, and optimization, as well as determination of compound off-target activities. Taking advantage of robotic systems to maintain cell culture and perform cell proliferation assays would greatly increase productivity and efficiency. Here we describe the establishment of automated systems for high-throughput cell proliferation assays in a panel of 13 human tumor cell lines. These cell lines were selected from various types of human tumors containing a broad range of well-characterized mutations in multiple cellular signaling pathways. Standard procedures for cell culture and assay performance were developed and optimized in each cell line. Moreover, in-house developed software (i.e., Toolset, Curvemaster, and Biobars) was applied to analyze the data and generate data reports. Using tool compounds, we have shown that results obtained through this panel exhibit high reproducibility over a long period. Furthermore, we have demonstrated that this panel can be used to identify sensitive and insensitive cell lines for specific cancer targets, to drive cellular structure-activity relationships, and to profile compound off-target activities. All those efforts are important for cancer drug discovery lead optimization.

In recent years, the increased use of cell-based functional assays for G protein-coupled receptors in high-throughput screening has enabled the design of robust assays to identify allosteric modulators (AMs) in addition to the more traditional orthosteric agonists and antagonists. In this article, the authors describe a screening format able to identify all ligand types using a triple-add assay that measures changes in cytosolic calcium concentration with three separate additions and reads in the same assay plate. This triple-add assay captures more small molecule ligand types than previously described assay formats without a significant increase in screening cost. Finally, the customizability of the triple-add assay to suit the needs of various AM screening programs is demonstrated.

The drug discovery process is dependent on the ability of screening efforts to identify and optimize lead compounds with therapeutic potential. Because G-protein-coupled receptors (GPCRs) account for the most "druggable" targets, the development of high-throughput, low-cost, and high-density GPCR assays to accommodate the increasing size of compound collections is significant. In this study, we report the application of an advanced LEADseeker robotic platform equipped with customized IT solutions for rapid data transfer to reduce assay cycles times for support of GPCR panel screening. The advantages of assay throughput, format, automation design, data management flow, and data reproducibility are discussed in terms of gains in productivity for lead optimization. The GPCR robotic platform demonstrates how automation technology can leverage traditional drug discovery processes by providing consistent and reliable data packages to expedite lead optimization efforts.

Low-volume dispensing of neat dimethyl sulfoxide (DMSO) into plate-based assays conserves compound, assay reagents, and intermediate dilution plate cost and, as we demonstrate here, significantly improves structure-activity relationship resolution. Acoustic dispensing of DMSO solutions into standard volume 384W plates yielded inconsistent results in studies with 2 cell lines because of apparent effects on the integrity of the cell monolayer (increased intracellular Ca⁺⁺ levels as indicated by elevated basal dye fluorescence after acoustic transfer). PocketTip-mediated transfer was successful at increasing apparent potency on a more consistent basis. Notably, the correlation coefficient among fluorescence imaging plate reader (FLIPR):electrophysiology (EP) across a representative ~125 compound collection was increased ~5× via conversion to a PocketTip direct dispensation, indicating a triage assay more predictive of activity in the decisional patch-clamp assay. Very importantly, the EP-benchmarked false-negative rate as measured by compounds with FLIPR EC₅₀ more than the highest concentration tested fell from >11% to 5% assay-wide, and the relative FLIPR:EP rank-order fidelity increased from 55% to 78%. Elimination of the aqueous intermediate step provided additional benefits, including reduced assay cost, decreased cycle time, and reduced wet compound consumption rate. Direct DMSO dispensing has broad applicability to cell-based functional assays of multiple varieties, especially in cases where limit solubility in assay buffer is a recognized impediment to maximizing interassay connectivity.