G3BP1/2

FAZ-3532 (G3Ia) and G3Ib disrupt in vitro condensation of RNA, G3BP1, and caprin 1.
Preincubation with FAZ-3532 (G3Ia) or G3Ib prevents the formation of stress granules in cells.
Treatment with FAZ-3532 (G3Ia) and G3Ib rapidly dissolves preformed stress granules.
Treatment with G3I compounds does not influence the rate of translation in cells.
Treatment with FAZ-3532 (G3Ia) prevents the formation of stress granules and dissolves preformed stress granules in human iPSC-derived neurons.
Treatment with FAZ-3532 (G3Ia) or G3Ib dissolves stress granules formed in response to expression of a disease-causing VCP mutant.
Treatment with FAZ-3532 (G3Ia) results in the removal of G3BP1 from stress granules formed in response to the expression of disease-causing FUS mutant.[1]

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Stress granule formation is triggered by the release of mRNAs from polysomes and is promoted by the action of the RNA-binding proteins G3BP1/2. 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, G3BP inhibitor a and b (FAZ-3532 (G3Ia) and G3Ib), designed to bind to a specific pocket in G3BP1/2 that is targeted by viral inhibitors of G3BP1/2 function. In addition to disrupting the 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. These effects are consistent across multiple cell types and a variety of initiating stressors. Thus, these compounds represent powerful tools to probe the biology of stress granules and hold promise for therapeutic interventions designed to modulate stress granule formation.[1]

Imaging of mutation-induced granules and analysis[1]
U2OS cells with knock-in of G3BP1-tdTomato (Yang et al., 2020) were seeded into 96-well plates. Either GFP-VCP-A232E or GFP-FUS-R495X were transfected into cells using ViaFect transfection reagent 24 h prior to the start of the experiment. For samples to be treated with compounds FAZ-3532 (G3Ia) or G3Ia′, Hoechst (1:5,000) was added and incubated for 30 min prior to the start of the experiment, washed once with PBS, and the media was changed to 100 μl FluoroBrite DMEM media upon starting the experiment. Imaging of plates was performed on a Cytation C10 spinning disk confocal with a Hamamatsu Orca-Flash 4.0 camera using Gen5 software (version 3.11). Imaging was performed through an Olympus Plan Apo 40× 0.6 NA dry objective with an adjustable collar set to 0.5 μm thickness. Laser autofocus prior to imaging the tdTomato channel was utilized for each image. The temperature was maintained at 37°C on the instrument and the cells were supplied with 5% CO2. A 7 × 7 tilescan was taken at the center of each well, capturing the tdTomato and GFP channels (and Hoechst when present). After completing the tilescan, 100 μl FluoroBrite with 100 μM (2×) G3I compound (vortexed for 5 s) was added to each well and incubated for 20 min before starting another round of imaging in the same locations as the first round. Samples treated with G3Ib or G3Ib′ were analyzed manually, scoring each cell with spontaneous granules for the presence or absence of granules following treatment with the compound. For experiments in which G3I compounds were pretreated prior to transfection, 50 μM of indicated compound or vehicle control were added to U2OS cells 20 min prior to the addition of the transfection reagent. After 24 h of transfection, the cells were washed with PBS, fixed in 4% formaldehyde, and incubated with Hoechst (1:5,000) in PBS for 30 min prior to the start of the experiment, washed once with PBS, and at room temperature imaging on the Cytation C10 spinning disk confocal was performed as described above.[1]
Samples treated with FAZ-3532 (G3Ia) or G3Ia′ were analyzed using an automated pipeline. Segmentation was performed in both ilastik and Cellpose 2.0. Nuclear segmentation using the Hoechst channel and granule segmentation in both GFP and tdTomato channels was performed via pixel classification in ilastik using small cropped portions of the tilescan as the training dataset for each. Individual cell segmentation was performed in Cellpose 2.0 by inputting a merged RGB of the Hoechst and G3BP1-tdTomato channels. The Cellpose output was then eroded by one pixel and set as a binary mask in Fiji. The original three raw channels, three output masks from ilastik, and the one mask generated from Cellpose were all inputted into CellProfiler to generate per-cell measurements for granules as defined by GFP or tdTomato.

[1].Identification of small molecule inhibitors of G3BP-driven stress granule formation. J Cell Biol. 2024 Mar 4;223(3):e202308083.

The use of FAZ-3532 （G3Ia） and G3Ib to target the NTF2L domain of G3BP1/2 has several potential applications. Primarily, these compounds will provide a tool for researchers to manipulate G3BP1/2-dependent stress granule formation and identify specific functions of stress granules in cells. Unlike many other stress granule inhibitors such as cycloheximide (Mollet et al., 2008) that lead to global inhibition of translation and are highly toxic, FAZ-3532 （G3Ia） and G3Ib are designed to be specific in their inhibition of protein binding within the NTF2L domain of G3BP1/2. FAZ-3532 （G3Ia） and G3Ib impair condensate formation in a highly tractable manner, do not appear to induce cellular toxicity or growth phenotypes (Fig. 2), and are versatile among different cell types. While NTF2L is also the dimerization domain of G3BP1/2 proteins, G3Ia and G3Ib bind to an interaction surface that is on the opposite face of the dimerization surface, thereby leaving dimerization intact (Fig. 1 D). Thus, FAZ-3532 （G3Ia） and G3Ib target only the protein–protein interaction domain of G3BP1/2 and specifically block G3BP1/2 condensate formation while leaving the dimerization and RNA-binding capabilities of G3BP1/2 unaltered. This approach also presents a significant advantage over genetic models in which G3BP1 and G3BP2 are knocked out, resulting in a complete loss of function of these proteins along with potential alterations in the expression of other stress granule proteins. Stress granules have been implicated in several disease states, including neurodegeneration and cancer (Protter and Parker, 2016). These compounds could be utilized in disease models to block stress granule formation or potentially dissolve disease-initiated granules, allowing for a better understanding of the roles of these condensates in disease initiation and progression. Consistent with this possibility, we observed that FAZ-3532 （G3Ia） and G3Ib can induce the disassembly of aberrant stress granules triggered by the expression of a pathogenic VCP A232E mutation that causes multisystem proteinopathy (Fig. 5). Additionally, G3Ia and G3Ib can be used to probe the immunological role of stress granules, as G3BP1-mediated stress granules are known to form in response to infection with numerous viral RNAs (Jayabalan et al., 2023). Finally, these compounds can be used to define whether the NTF2L domain-mediated interactions of G3BP1/2 play a role in the normal function of cells outside of their ability to drive condensation. Such studies could lead to the discovery of novel stress granule-independent functions of G3BP1, G3BP2, or their interactors in normal cell biology. In summary, FAZ-3532 （G3Ia） and G3Ib are potent inhibitors of binding to the NTF2L domain of G3BP1/2 that are highly specific, easy to synthesize, and highly effective across multiple cell types and stressors, providing a valuable new tool in the study of condensate biology.

C38H56FN5O7

713.88

713.41637

170459244

White to off-white solid powder

4

OULWYQHHPPQJBM-AZWKTQJSSA-N

InChI=1S/C38H56FN5O7/c1-24(27-18-14-11-15-19-27)30(42-35(48)31(38(6,7)50)43-33(46)28(39)22-37(3,4)5)34(47)40-25(2)32(45)41-29(20-21-51-9)36(49)44(8)23-26-16-12-10-13-17-26/h10-19,24-25,28-31,50H,20-23H2,1-9H3,(H,40,47)(H,41,45)(H,42,48)(H,43,46)/t24-,25+,28-,29-,30-,31+/m0/s1

(2S)-N-[(2S)-1-[[(2S,3S)-1-[[(2R)-1-[[(2S)-1-[benzyl(methyl)amino]-4-methoxy-1-oxobutan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxo-3-phenylbutan-2-yl]amino]-3-hydroxy-3-methyl-1-oxobutan-2-yl]-2-fluoro-4,4-dimethylpentanamide

FAZ-3532; FAZ3532

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Typically soluble in DMSO (e.g. 10 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)