(A) Fluorescence polarization (FP) assays showing binding of ISRIB to human eIF2B. Left panel: a plot of the FP signal arising from fluorescein-labeled ISRIB analog (AAA2-101) (2.5 nM) as a function of the concentration of eIF2B in the sample. Right panel: a plot of the relative FP signal arising from samples with fluorescein-labeled AAA2-101 (2.5 nM) bound to purified human eIF2B (30 nM) in the presence of the indicated concentration of unlabeled trans-ISRIB introduced as a competitor. Concentrations of eIF2B and ISRIB on respective plots are represented on a log₁₀ scale. Curve fitting and EC₅₀ was generated using agonist vs. response function on GraphPad Prism, shown are values of three independently-acquired measurements.
(B) Representative views of the cryo-EM map of the ISRIB-bound decameric human eIF2B complex. Density is colored according to the subunit architecture indicated in the cartoons: α – blue, β – cyan, δ – green, γ – gold, ε – pink, ISRIB - orange.
(C) Ribbon representation of ISRIB-bound human eIF2B “central” view of the (βδ)₂ dimer interface with a single molecule of ISRIB.
(D) Close-up of the “central” view showing the ISRIB binding site. An ISRIB molecule is docked into the cavity at the (βδ)₂ dimer interface. Residues contacting ISRIB in the central part of the pocket from the β (blue) and δ (green) subunits are indicated. ISRIB is represented in orange sticks.

(A) Histograms of the ISR-responsive CHOP::GFP fluorescent reporter activity, induced by histidinol (HIS⁺, 0.5 mM) in ISRIB-sensitive (ISRIB^SEN) (left panels) and ISRIB-resistant (ISRIB^RES) (right panels) pools of CHO-K1 cells, selected for their responsiveness to ISRIB (200 nM), following CRISPR/Cas9-induced random mutagenesis of the indicated codon of Eif2b2.
(B) Bar graph of the distribution of residues identified at the indicated positions of mutagenized Eif2b2, analyzed by the next generation sequencing (NGS). Shown is the number of sequenced reads in ISRIB^SEN pools (orange bars) or ISRIB^RES pools (plum bars) encoding each amino acid (* - stop codon, X – ambiguous sequence).
(C) Graphs showing inhibition of the ISR-activated CHOP::GFP reporter (induced as in panel “A”) by ISRIB or two related analogs, compound AAA1-075B (075B) and compound AAA1-084 (084), in ISRIB^SEN (left) and ISRIB^RES (right) mutant pools of EIf2b2^H188X. Shown is a representative from three independent experiments for each of the compounds. Concentration of inhibitor is represented on a log₁₀ scale. Curve fitting and EC₅₀ was generated using agonist vs. response function on GraphPad Prism.

(A) Histograms of the ISR-responsive CHOP::GFP fluorescent reporter activity, induced by histidinol (HIS⁺, 0.5 mM) in ISRIB^RES, ISRIB^SEN, compound 075B^SEN and compound 084^SEN sub-pools, selected for their responsiveness to ISRIB or its analogs (2.5 µM) from a population of originally ISRIB^RES Eif2b2^H188X mutant cells.
(B) Bar graph of the distribution of residues identified at Eif2b2 codon 188 in phenotypically divergent pools of CHO-K1 cells. The number of sequenced reads in ISRIB^RES (plum), ISRIB^SEN (orange), compound 075B^SEN (blue) and compound 084^SEN (cyan) pools encoding each amino acid (* - stop codon, X – ambiguous) is plotted.
(C) Plot of the relative FP signal arising from samples with fluorescein-labeled AAA2-101 (2.5 nM) bound to purified hamster eIF2B (30 nM) in the presence of the indicated concentration of unlabeled ISRIB introduced as a competitor (represented on a log₁₀ scale). Shown is a representative of two independent experiments. The fitting curve and EC₅₀ was generated using “agonist vs. response” function on GraphPad Prism.
(D) A plot of the FP signal arising from fluorescein-labeled AAA2-101 (2.5 nM) as a function of the concentration of wildtype (wt) or mutant eIF2B (δ^L180F or β^H188K) in the sample. Shown are mean ± SD (n=3). Concentrations of eIF2B are represented on a log₁₀ scale.