Identification of small molecule inhibitors of G3BP-driven stress granule formation

Abstract

Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the paralogs G3BP1 and G3BP2. G3BP1/2 proteins bind mRNAs and thereby promote the condensation of mRNPs into stress granules. Stress granules have been implicated in several disease states, including cancer and neurodegeneration. Consequently, compounds that limit stress granule formation or promote their dissolution have potential as both experimental tools and novel therapeutics. Herein, we describe two small molecules, referred to as G3BP inhibitor a and b (G3Ia and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is known to be targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting co-condensation of RNA, G3BP1, and caprin 1 in vitro, these compounds inhibit stress granule formation in cells treated prior to or concurrent with stress, and dissolve pre-existing stress granules when added to cells after stress granule formation. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent ideal tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.

Figure 1.. Lead compounds G3Ia and G3Ib bind with high affinity to the NTF2L nsP3 binding pocket of G3BP1.

(A) Lead compounds FAZ-3532 (G3Ia) and FAZ-3780 (G3Ib) along with respective enantiomer controls FAZ-3861 (G3Ia’) and FAZ-3852 (G3Ib’). (B) Representative double-reference subtracted sensorgrams of compounds binding to sensor-immobilized human G3BP1. Compounds were tested by 1/2 dilution with top concentration of 50 μM (G3Ia’), 50 μM (G3Ib’), 10 μM (G3Ia), and 2 μM (G3Ib). Marks on each curve indicate the time span at which equilibrium binding was measured in order to estimate the equilibrium dissociation constant using a 1:1 Langmuir binding model. (C) Percent inhibition calculated using the peptide displacement assay at indicated doses of G3I compounds. Error bars represent mean ± SD, n = 2 replicates per dose. (D) Crystal structure showing the interaction of G3Ia with the nsP3 binding pocket in the NTF2L domain of G3BP1. The NTF2L domain of G3BP1 (light green cartoon model) crystallized in the presence of G3Ia (yellow sticks), with six copies in the asymmetric unit and copy A shown above. All copies were compound bound, although only half had full compound density. The other three were incomplete in either the ether group (1) or terminal phenylalanine (2) highlighting their flexibility. Tert-butyl (3) functions as a space-filling moiety, maximizing the hydrophobicity of the subpocket lined by V11 and F124. An indirect water-mediated backbone interaction with Q18 is present in four of six copies, including copy A above (large red ball). (E) Summary characteristics of the four G3I compounds compared to the nsP3 25-mer peptide. PSA, polar surface area.

Figure 2.. G3Ia and G3Ib disrupt in vitro condensation of RNA, G3BP1, and caprin 1.

(A) 1.5 μM G3BP1, 1.5 μM caprin 1, and 20 ng/μL total RNA were co-incubated in a three-component system and co-condensation was assessed in the presence of increasing concentrations of G3I compounds. The percent inhibition of G3BP1-GFP in vitro phase separation is shown. Error bars represent mean ± SD. (B) 20 μM purified G3BP1 and 100 ng genomic RNA were co-incubated in a two-component system and condensation was assessed in the presence of indicated doses of G3Ib or vehicle control. Condensate formation by G3BP1 and RNA was unaffected by the addition of G3Ib. (C) U2OS cells were treated with indicated concentrations of compounds for 24 h and inhibition of growth was measured by monitoring ATP levels, read out through a luciferase signal. N=2, both replicates are plotted.

Figure 3.. Pre-incubation with G3Ia or G3Ib prevents the formation of stress granules in living cells.

(A) Schematic showing the pre-incubation paradigm used in panels B-C. Indicated doses of compound were added to cells for 20 min, followed by exposure to 500 μM NaAsO₂ stress and live cell imaging to monitor stress granule formation. (B) Representative images of G3BP1-GFP signal in U2OS cells after 10 min or 20 min of oxidative stress. Scale bars, 40 μm. (C) Quantification of cells as in (B) showing the percentage of stress granule area per cell. (D) Schematic showing the pre-incubation paradigm used in panels E-F. 50 μM of indicated compounds was added to cells for 20 min, followed by exposure to 43°C heat shock for 30 min. Live cell imaging was used to monitor stress granule formation. (E) Representative images of G3BP1-GFP signal in U2OS cells 15 min post heat shock. Scale bar, 40 μm. (F) Quantification of cells as in (E) showing the percentage of stress granule area per cell throughout heat shock (43°C, 30 min) and recovery (37°C, 43 min). Error bars represent mean ± SEM in all graphs.

Figure 4.. Treatment with G3Ia and G3Ib rapidly dissolves pre-formed stress granules.

(A) Representative images of G3BP1-GFP signal in U2OS cells following induction of stress by 250 μM NaAsO₂. Images are shown at 30 min after induction of stress (immediately before addition of compound), 32 min after induction of stress (2 min after addition of 50 μM G3I compound), and 40 min after induction of stress (10 min after addition of 50 μM G3I compound). Scale bar, 40 μm. (B) Quantification of cells as in (A) showing the percentage of stress granule area per cell. (C) Quantification of the percentage of stress granule area per cell throughout heat shock (43°C, 30 min) and recovery (37°C) from U2OS cells stably expressing G3BP1-GFP. Cells were treated with 50 μM G3I compound 25 min after the induction of heat shock. (D) Schematic showing the experimental paradigm used in panels E-F. U2OS cells stably expressing G3BP1-GFP were exposed to 250 μM NaAsO₂ for 30 min followed by the addition of 50 μM G3I compound. Cells were fixed and stained 5 min after compound was added. (E-F) Shown are representative images of immunofluorescence staining of additional stress granule markers (eIF3η, PABPC1, FXR1). Scale bars, 20 μm. Error bars represent mean ± SEM in all graphs.

Figure 5.. Treatment with G3I compounds modifies stress granules formed in response to expression of disease-causing mutant proteins.

(A) U2OS cells with TdTomato-tagged endogenous G3BP1 (red) were transfected with VCP A232E (green) for 24 h, then treated with 50 μM G3I compound for 30 minutes. Shown are representative images of cells before (left) and after (right) addition of G3Ia or G3Ia’. Scale bar, 10 μm. (B) Quantification of cells as in (A) showing the percentage of stress granule as assessed by TdTomato imaging. Automated puncta tracking was used for G3Ia; manual blinded cell tracking was used for G3Ib. (C) U2OS cells with TdTomato-tagged endogenous G3BP1 (red) were transfected with FUS R495X (green) for 24 h, then treated with 50 μM G3I compound for 30 minutes. Shown are representative images of cells before (left) and after (right) addition of G3Ia or G3Ia’. Scale bar, 10 μm. (D) Quantification of cells as in (C) showing the percentage of puncta dissolution for G3BP1-positive and FUS R495X-positive puncta.