Diverse stress signals converge at a single phosphorylation event at serine 51 of eIF2α, kinases of which are PERK, GCN2, PKR and HRI and this four kinases form a collection of signaling pathways termed the “integrated stress response” (ISR) as they converge on the same molecular event. The ternary complex (eIF2-GTP-Met-tRNAi) can associate with the 40S ribosomal subunit forming the 43S pre-initiation complex (PIC) that scans the 5’ UTR of mRNAs to select the initiating AUG codon. ISRIB as a potent ISR (integrated stress response) inhibitor, which functions downstream of all eIF2α kinases. The ISRIB trans-Isomer showed effect on inhibition of the ATF4 luciferase reporter in HEK293T cells treated with 2 µg/ml of tunicamycin to induce ER stress with IC50 value of 5nM. Treatment with 200nM ISRIB trans-Isomer blocked production of endogenous ATF4 induced by tunicamycin or thapsigargin, the targets of ATF4 including CHOP and GADD34, as well as blocked ATF4 induction after activation of either GCN2 or HRI, but did not affect XBP1 mRNA splicing, XBP1s production or PERK phosphorylation in HEK293T cells, suggesting that ISRIB specifcally blocks signaling of the PERK-branch of the UPR but did not impair PERK phosphorylation or activation of the ATF6-branch of the UPR. ISRIB did not block eIF2α phosphorylation under ER stress-inducing conditions, even increased the level of eIF2α phosphorylation at 120 min after tunicamycin addition, suggesting that ISRIB may function on effects downstream of PERK and eIF2α phosphorylation, but not PERK or eIF2α phosphorylation itself. A further experiment showed that ISRIB trans-Isomer can help maintain high PIC levels even when eIF2α is phosphorylated and exert its function by maintaining elevated ternary complex (eIF2-GTP-Met-tRNAi) levels. ISRIB addition caused a strong synergistic effect on ER-stressed cells by tunicamycin, reducing colony number and size due to apoptosis signifcantly more than ER-stress alone in HEK293T cells, while ISRIB itself did not affect cell viability alone. ISRIB is brain permeable. Animal dosed with ISRIB showed enhancement on both hippocampusdependent spatial learning and hippocampus-dependent contextual fear conditioning[1].
作用机制
ISRIB trans-Isomer can exert its function by maintaining elevated ternary complex (eIF2-GTP-Met-tRNAi) levels. Notice that ER stress remains unmitigated in ISRIB-treated cells is supported by sustained activation of all three UPR sensors including PERK, IRE1 and ATF6.[1]
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