Pharmacologic IRE1/XBP1s activation confers targeted ER proteostasis reprogramming

Abstract

Activation of the IRE1/XBP1s signaling arm of the unfolded protein response (UPR) is a promising strategy to correct defects in endoplasmic reticulum (ER) proteostasis implicated in diverse diseases. However, no pharmacologic activators of this pathway identified to date are suitable for ER proteostasis remodeling through selective activation of IRE1/XBP1s signaling. Here, we use high-throughput screening to identify non-toxic compounds that induce ER proteostasis remodeling through IRE1/XBP1s activation. We employ transcriptional profiling to stringently confirm that our prioritized compounds selectively activate IRE1/XBP1s signaling without activating other cellular stress-responsive signaling pathways. Furthermore, we demonstrate that our compounds improve ER proteostasis of destabilized variants of amyloid precursor protein (APP) through an IRE1-dependent mechanism and reduce APP-associated mitochondrial toxicity in cellular models. These results establish highly selective IRE1/XBP1s activating compounds that can be widely employed to define the functional importance of IRE1/XBP1s activity for ER proteostasis regulation in the context of health and disease.

Small molecule strategies to harness the unfolded protein response: where do we go from here?

“…2G). Importantly, these compounds were non-toxic in HEK293 TREX cells (IC50 > 3µM) (85). The selectivity of these compounds for the IRE1-XBP1s pathway was further defined by RNA-seq transcriptional profiling, confirming that these compounds do not activate other arms of the UPR or other stressresponsive signaling pathways (85).…”

“…This screening strategy prioritized transcriptional selectivity to identify compounds that show preferential activation of IRE1/XBP1s over other arms of the UPR or other stressresponsive signaling pathways. This HTS used an IRE1-dependent XBP1 splicing luciferase reporter to screen a >650k library to identify IRE1 activating compounds (85). Hits were then counterscreened against a luciferase reporter for the ATF6 UPR signaling pathway to identify compounds which preferentially activate IRE1/XBP1s signaling over other arms of the UPR.…”

“…Importantly, these compounds were non-toxic in HEK293 TREX cells (IC50 > 3µM) (85). The selectivity of these compounds for the IRE1-XBP1s pathway was further defined by RNA-seq transcriptional profiling, confirming that these compounds do not activate other arms of the UPR or other stressresponsive signaling pathways (85). Importantly, these compounds increase IRE1/XBP1s signaling without inhibiting kinase activity, indicating that these compounds activate IRE1 through a mechanism distinct from the above-mentioned Type I kinase inhibitors (85).…”

“…Importantly, these compounds increase IRE1/XBP1s signaling without inhibiting kinase activity, indicating that these compounds activate IRE1 through a mechanism distinct from the above-mentioned Type I kinase inhibitors (85). While the mechanism of action for these compounds as well as in vivo efficacy remains to be established, these compounds have already proven useful for demonstrating the potential for pharmacologic IRE1 activation to reduce the production and toxicity of amyloid precursor protein (APP) proteolytic fragments in cell culture models (85).…”

“…Over the past 5 years multiple different strategies have been developed to probe the selectivity of compounds for specific stress-responsive transcriptional signaling pathways, including the three arms of the UPR. For example, the integration of reporters for multiple different UPR signaling pathways into the screening pipeline has proven useful in identifying compound selectivity for a specific pathway in a high-throughput format (85,205). Similarly, incorporating cell-based toxicity screening early in a HTS pipeline provides a useful approach to remove compounds that are toxic through either on-target or off-target pathways (85,205), although this toxicity could still emerge when compounds are applied to other cell models.…”