The endoplasmic reticulum (ER) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one-third of cellular proteins, and is thus inextricably linked to the maintenance of cellular homeostasis and the fine balance between health and disease. Specific ER stress signalling pathways, collectively known as the unfolded protein response (UPR), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand and the UPR initially aims to restore ER homeostasis and normal cellular functions. However, if this fails, then the UPR triggers cell death. In this review, we provide a UPR signalling-centric view of ER functions, from the ER's discovery to the latest advancements in the understanding of ER and UPR biology. Our review provides a synthesis of intracellular ER signalling revolving around proteostasis and the UPR, its impact on other organelles and cellular behaviour, its multifaceted and dynamic response to stress and its role in physiology, before finally exploring the potential exploitation of this knowledge to tackle unresolved biological questions and address unmet biomedical needs. Thus, we provide an integrated and global view of existing literature on ER signalling pathways and their use for therapeutic purposes.

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Inositol‐requiring enzyme 1 (IRE1) is a bifunctional serine/threonine kinase and endoribonuclease that is a major mediator of the unfolded protein response (UPR) during endoplasmic reticulum (ER) stress. Tumour cells experience ER stress due to adverse environmental cues such as hypoxia or nutrient shortage and high metabolic/protein‐folding demand. To cope with those stresses, cancer cells utilise IRE1 signalling as an adaptive mechanism. Here, we report the discovery of the FDA‐approved compounds methotrexate, cefoperazone, folinic acid and fludarabine phosphate as IRE1 inhibitors. These were identified through a structural exploration of the IRE1 kinase domain using IRE1 peptide fragment docking and further optimisation and pharmacophore development. The inhibitors were verified to have an impact on IRE1 activity in vitro and were tested for their ability to sensitise human cell models of glioblastoma multiforme (GBM) to chemotherapy. We show that all molecules identified sensitise glioblastoma cells to the standard‐of‐care chemotherapy temozolomide (TMZ).

IRE1 is an endoplasmic reticulum (ER) bound transmembrane bifunctional kinase and endoribonuclease protein crucial for the unfolded protein response (UPR) signaling pathway. Upon ER stress, IRE1 homodimerizes, oligomerizes and autophosphorylates resulting in endoribonuclease activity responsible for excision of a 26 nucleotide intron from the X-box binding protein 1 (XBP1) mRNA. This unique splicing mechanism results in activation of the XBP1s transcription factor to specifically restore ER stress. Small molecules targeting the reactive lysine residue (Lys907) in IRE1α’s RNase domain have been shown to inhibit the cleavage of XBP1 mRNA. Crystal structures of murine IRE1 in complex with covalently bound hydroxyl aryl aldehyde (HAA) inhibitors show that these molecules form hydrophobic interactions with His910 and Phe889, a hydrogen bond with Tyr892 and an indispensable Schiff-base with Lys907. The availability of such data prompted interest in exploring structure-based drug design as a strategy to develop new covalently binding ligands. We extensively evaluated conventional and covalent docking for drug discovery targeting the catalytic site of the RNase domain. The results indicate that neither computational approach is fully successful in the current case, and we highlight herein the potential and limitations of the methods for the design of novel IRE1 RNase binders.

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