Deconvoluting Stress-Responsive Proteostasis Signaling Pathways for Pharmacologic Activation Using Targeted RNA Sequencing

Cellular
proteostasis is maintained by stress-responsive signaling pathways
such as the heat shock response (HSR), the oxidative stress response
(OSR), and the unfolded protein response (UPR). Activation of these
pathways results in the transcriptional upregulation of select subsets
of stress-responsive genes that restore proteostasis and adapt cellular
physiology to promote recovery following various types of acute insult.
The capacity for these pathways to regulate cellular proteostasis
makes them attractive therapeutic targets for correcting proteostasis
defects associated with diverse diseases. High-throughput screening
(HTS) using cell-based reporter assays is highly effective for identifying
putative activators of stress-responsive signaling pathways. However,
the development of these compounds is hampered by the lack of medium-throughput
assays to define compound potency and selectivity for a given pathway.
Here, we describe a targeted RNA sequencing (RNAseq) assay that allows
cost-effective, medium-throughput screening of stress-responsive signaling
pathway activation. We demonstrate that this assay allows deconvolution
of stress-responsive signaling activated by chemical genetic or pharmacologic
agents. Furthermore, we use this assay to define the selectivity of
putative OSR and HSR activating compounds previously identified by
HTS. Our results demonstrate the potential for integrating this adaptable
targeted RNAseq assay into screening programs focused on developing
pharmacologic activators of stress-responsive signaling pathways.