The endoplasmic reticulum (ER) is involved in Ca2+ signaling and protein folding. ER Ca2+ depletion and accumulation of unfolded proteins activate the molecular chaperone GRP78 (glucose-regulated protein 78) which in turn triggers the ER stress response (ERSR) pathway aimed to restore ER homeostasis. Failure to adapt to stress, however, results in apoptosis. We and others have shown that malignant cells are more susceptible to ERSR-induced apoptosis than their normal counterparts, implicating the ERSR as a potential target for cancer therapeutics. Predicated on these findings, we developed an assay that uses a GRP78 biosensor to identify small molecule activators of ERSR in glioma cells. We performed a quantitative high-throughput screen (qHTS) against a collection of ~425,000 compounds and a comprehensive panel of orthogonal secondary assays was formulated for stringent compound validation. We identified novel activators of ERSR, including a compound with a 2,9-diazaspiro[5.5]undecane core, which depletes intracellular Ca2+ stores and induces apoptosis-mediated cell death in several cancer cell lines, including patient-derived and 3D cultures of glioma cells. This study demonstrates that our screening platform enables the identification and profiling of ERSR inducers with cytotoxic activity and advocates for characterization of these compound in in vivo models.

INTRODUCTION: High-resolution microscopy using fluorescent probes is a powerful tool to investigate individual cell structure and function, cell subpopulations and mechanisms underlying cellular responses to drugs. Additionally, responses to drugs more closely resemble those seen in vivo when cells are physically connected in three-dimensional (3D) systems (either 3D cell cultures or whole organisms), as opposed to traditional monolayer cultures. Combined, the use of imaging-based 3D models in the early stages of drug development has the potential to generate biologically relevant data that will increase the likelihood of success for drug candidates in human studies. AREAS COVERED: The authors discuss current methods for the culturing of cells in 3D as well as approaches for the imaging of whole-animal models and 3D cultures that are amenable to high-throughput settings and could be implemented to support drug discovery campaigns. Furthermore, they provide critical considerations when discussing imaging these 3D systems for high-throughput chemical screenings. EXPERT OPINION: Despite widespread understanding of the limitations imposed by the two-dimensional versus the 3D cellular paradigm, imaging-based drug screening of 3D cellular models is still limited, with only a few screens found in the literature. Image acquisition in high throughput, accurate interpretation of fluorescent signal, and uptake of staining reagents can be challenging, as the samples are in essence large aggregates of cells. The authors recognize these shortcomings that need to be overcome before the field can accelerate the utilization of these technologies in large-scale chemical screens.

Copy number variation resulting in excess PMP22 protein causes the peripheral neuropathy Charcot-Marie-Tooth disease, type 1A. To broadly interrogate chemically sensitive transcriptional pathways controlling PMP22 protein levels, we used the targeting precision of TALEN-mediated genome editing to embed reporters within the genetic locus harboring the Peripheral Myelin Protein 22 (Pmp22) gene. Using a Schwann cell line with constitutively high endogenous levels of Pmp22, we obtained allelic insertion of secreted bioluminescent reporters with sufficient signal to enable a 1536-well assay. Our findings from the quantitative high-throughput screening (qHTS) of several thousand drugs and clinically investigated compounds using this assay design both overlapped and expanded results from a previous assay using a randomly inserted reporter gene controlled by a single regulatory element of the Pmp22 gene. A key difference was the identification of a kinase-controlled inhibitory pathway of Pmp22 transcription revealed by the activity of the Protein kinase C (PKC)-modulator bryostatin.

Argonaute2 (Ago2) protein and associated microRNAs (miRNAs) or small interfering RNAs (siRNAs) form the RNA-induced silencing complex (RISC) for target messenger RNA cleavage and post-transcriptional gene silencing. Although Ago2 is essential for RISC activity, the mechanism of RISC assembly is not well understood, and factors controlling Ago2 protein expression are largely unknown. A role for the Hsc70/Hsp90 chaperone complex in loading small RNA duplexes into the RISC has been demonstrated in cell extracts, and unloaded Ago2 is unstable and degraded by the lysosome in mammalian cells. Here we identify the co-chaperones Fkbp4 and Fkbp5 as Ago2-associated proteins in mouse embryonic stem cells. Pharmacological inhibition of this interaction using FK506 or siRNA-mediated Fkbp4/5 depletion leads to decreased Ago2 protein levels. We find FK506 treatment inhibits, whereas Fkbp4/5 overexpression promotes, miRNA-mediated stabilization of Ago2 expression. Simultaneous treatment with a lysosome inhibitor revealed the accumulation of unloaded Ago2 complexes in FK506-treated cells. We find that, consistent with unloaded miRNAs being unstable, FK506 treatment also affects miRNA abundance, particularly nascent miRNAs. Our results support a role for Fkbp4/5 in RISC assembly.