Targeting Phosphopeptide Recognition by the Human BRCA1 Tandem BRCT Domain to Interrupt BRCA1-Dependent Signaling.

Intracellular signals triggered by DNA breakage flow through proteins containing BRCT (BRCA1 C-terminal) domains. This family, comprising 23 conserved phosphopeptide-binding modules in man, is inaccessible to small-molecule chemical inhibitors. Here, we develop Bractoppin, a drug-like inhibitor of phosphopeptide recognition by the human BRCA1 tandem (t)BRCT domain, which selectively inhibits substrate binding with nanomolar potency in vitro. Structure-activity exploration suggests that Bractoppin engages BRCA1 tBRCT residues recognizing pSer in the consensus motif, pSer-Pro-Thr-Phe, plus an abutting hydrophobic pocket that is distinct in structurally related BRCT domains, conferring selectivity. In cells, Bractoppin inhibits substrate recognition detected by Förster resonance energy transfer, and diminishes BRCA1 recruitment to DNA breaks, in turn suppressing damage-induced G2 arrest and assembly of the recombinase, RAD51. But damage-induced MDC1 recruitment, single-stranded DNA (ssDNA) generation, and TOPBP1 recruitment remain unaffected. Thus, an inhibitor of phosphopeptide recognition selectively interrupts BRCA1 tBRCT-dependent signals evoked by DNA damage

Endoplasmic Reticulum Stress signalling - from basic mechanisms to clinical applications

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

A rational computer-aided drug discovery approach to target IRE1 and PERK: Insights into structural dynamics and selectivity

Cells constantly monitor the number of misfolded proteins they accumulate. The accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers an evolutionarily conserved signaling pathway called the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) are the main UPR signal transducers. IRE1α and PERK were characterized using a chemical biology approach. Primarily, computational analysis of druggable sites of IRE1α and PERK was carried out. We performed docking studies to understand the selectivity of PERK kinase inhibitors. Additionally, KIT was identified as a common non-specific target of UPR kinase inhibitors kinase inhibiting RNase attenuator 6 (KIRA6) and GSK2606414 (GSK414). We analyzed the IRE1α RNase pocket for the suitability of structure-based drug screening. Further, we looked at how an IRE1α kinase inhibitor impacted the different dimer forms using in silico methods. Finally, virtual screening was performed for the IRE1α kinase active site. Novel lead compound NUIG10 was identified and validated in in vitro direct binding assay and RNase activity assay. Collectively, we highlighted various aspects of IRE1α and PERK drug discovery. In particular, our approaches provided structural insights into hit-identification and hit-to- lead optimization of novel IRE1 inhibitors.2024-05-1

A rational computer-aided drug discovery approach to target IRE1 and PERK: Insights into structural dynamics and selectivity

Cells constantly monitor the number of misfolded proteins they accumulate. The accumulation of misfolded proteins in the endoplasmic reticulum (ER) triggers an evolutionarily conserved signaling pathway called the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) are the main UPR signal transducers. IRE1α and PERK were characterized using a chemical biology approach. Primarily, computational analysis of druggable sites of IRE1α and PERK was carried out. We performed docking studies to understand the selectivity of PERK kinase inhibitors. Additionally, KIT was identified as a common non-specific target of UPR kinase inhibitors kinase inhibiting RNase attenuator 6 (KIRA6) and GSK2606414 (GSK414). We analyzed the IRE1α RNase pocket for the suitability of structure-based drug screening. Further, we looked at how an IRE1α kinase inhibitor impacted the different dimer forms using in silico methods. Finally, virtual screening was performed for the IRE1α kinase active site. Novel lead compound NUIG10 was identified and validated in in vitro direct binding assay and RNase activity assay. Collectively, we highlighted various aspects of IRE1α and PERK drug discovery. In particular, our approaches provided structural insights into hit-identification and hit-to- lead optimization of novel IRE1 inhibitors.2024-05-1

Peptidomimetic-based identification of FDA approved compounds inhibiting IRE1 activity

International audienceInositol 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 optimization 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)

The unfolded protein response modulators GSK2606414 and KIRA6 are potent KIT inhibitors

International audienceIRE1, PERK, and ATF6 are the three transducers of the mammalian canonical unfolded protein response (UPR). GSK2606414 is a potent inhibitor of PERK, while KIRA6 inhibits the kinase activity of IRE1. Both molecules are frequently used to probe the biological roles of the UPR in mammalian cells. In a direct binding assay, GSK2606414 bound to the cytoplasmic domain of KIT with dissociation constants (K) value of 664 ± 294 nM whereas KIRA6 showed a K value of 10.8 ± 2.9 µM. In silico docking studies confirmed a compact interaction of GSK2606414 and KIRA6 with KIT ATP binding pocket. In cultured cells, GSK2606414 inhibited KIT tyrosine kinase activity at nanomolar concentrations and in a PERK-independent manner. Moreover, in contrast to other KIT inhibitors, GSK2606414 enhanced KIT endocytosis and its lysosomal degradation. Although KIRA6 also inhibited KIT at nanomolar concentrations, it did not prompt KIT degradation, and rescued KIT from GSK2606414-mediated degradation. Consistent with KIT inhibition, nanomolar concentrations of GSK2606414 and KIRA6 were sufficient to induce cell death in a KIT signaling-dependent mast cell leukemia cell line. Our data show for the first time that KIT is a shared target for two seemingly unrelated UPR inhibitors at concentrations that overlap with PERK and IRE1 inhibition. Furthermore, these data underscore discrepancies between in vitro binding measurements of kinase inhibitors and inhibition of the tyrosine kinase receptors in living cells

Endoplasmic reticulum stress signalling - from basic mechanisms to clinical applications

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.status: publishe

Endoplasmic reticulum stress signalling - from basic mechanisms to clinical applications

International audienceThe 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