Formin Homology 2 (FH2) domains of Formin family proteins [1]

In U2OS cells, SMIFH2 (25 uM; 1–16 hours) causes dynamic cytoskeletal reorganization [1]. Protease-independent, SMIFH2 (25 μM) lowers p300, mDia2, and p53 levels [1]. By influencing cytoskeletal reorganization through a post-transcriptional, proteasome-independent mechanism, SMIFH2 lowers p53 expression and function [1].

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SMIFH2 induces dynamic remodeling of the actin cytoskeleton and microtubules in U2OS and HCT116 cells. Treatment for 1 hour reduces F-actin (phalloidin signal) and increases polymerized tubulin. At 2 hours, stress fibers and filopodia-like protrusions form. By 4-8 hours, lamellipodia and filopodia-like protrusions are observed. Microtubules are barely detectable at 2 hours, with recovery occurring after 16 hours. This results in alternated depolymerization-repolymerization cycles of actin and microtubules. [1]
- SMIFH2 causes scattering and a dramatic remodeling of the Golgi complex in U2OS and HCT116 cells, as evidenced by decreased Giantin staining intensity and scattered morphology starting at 1 hour, with near-complete signal loss at 4 hours and recovery by 16 hours. [1]
- SMIFH2 increases cell migration. Manually tracked U2OS cells treated with SMIFH2 showed increased net displacement and migration speed, particularly between 3 and 5 hours of treatment, correlating with lamellipodium formation. Directionality was not significantly affected. [1]
- SMIFH2 prevents or delays mitosis. In time-lapse experiments, entry into mitosis was a rare event in SMIFH2-treated U2OS cells compared to DMSO controls. [1]
- SMIFH2 downregulates the protein levels of mDia2, p53, and p300. This effect was observed in multiple cell lines (293T, A375, U2OS, MDA-MB-231, HCT116) and MEFs, independent of p53 status, expression levels, or transcriptional activity. The downregulation is post-transcriptional and proteasome-independent, as proteasome inhibitor Lactacystin failed to restore the levels of these proteins. Protein levels of mDia1, mDia3, actin, tubulin, and Giantin remained unchanged. [1]
- SMIFH2 attenuates p53 transcriptional activity. In 293T cells transfected with an HDM2-luciferase reporter, SMIFH2 treatment reduced luciferase activity, indicating decreased p53-mediated transcription. This effect was independent of mDia2. [1]
- p53 expression levels modulate the cellular response to SMIFH2. Side-by-side comparison of wild-type and p53-/- HCT116 cells showed that the decrease in F-actin and increase in microtubule signals at the 1-hour time point were linked to p53 expression. p53 knockdown U2OS cells showed more prominent cortical actin and stress fibers under growing conditions and were required for SMIFH2-induced lamellipodia protrusion. [1]

Western Blot Analysis[1]
Cell Types: 293T, A375, U2OS and MDA-MB-231 Cell
Tested Concentrations: 25 μM
Incubation Duration: 5 hrs (hours) for 293T, U2OS, MDA-MB-231 cells; 2.5 hrs (hours) for A375 cells
Experimental Results: mDia2, p53 and p300 protein levels were downregulated.

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Cell Culture and Treatments: U2OS, HCT116, 293T, A375, MDA-MB-231, and MEF cells were cultured in DMEM or DMEM/F12 medium with 10% FCS. Cells were treated with 25 µM SMIFH2 (or DMSO as control) for various durations (1, 2, 4, 8, 16 hours) unless otherwise specified. For high-dose experiments, SMIFH2 concentrations greater than 25 µM were used to observe cytotoxicity. [1]
- Immunofluorescence and Imaging: Cells plated on gelatin-coated coverslips were fixed with 4% paraformaldehyde in PIPES buffer, permeabilized, and stained with primary antibodies (anti-β-tubulin, anti-Giantin, anti-GM130, anti-TGN46) and secondary antibodies, along with TRITC-conjugated phalloidin for F-actin and DAPI for nuclei. Confocal images were acquired on a Leica TCS SP5. [1]
- Western Blotting: Total cell lysates were prepared in JS lysis buffer with protease and phosphatase inhibitors. Proteins were separated by SDS-PAGE and immunoblotted with antibodies against mDia1, mDia2, mDia3, p53, p300, p21, β-actin, β-tubulin, vinculin, and β-catenin. [1]
- Random Cell Migration Assay: U2OS cells were plated on gelatin-coated plates and allowed to adhere for 16 hours. Cells were imaged every 5 minutes in a humidified chamber (37°C, 5% CO2) in the presence of DMSO or 25 µM SMIFH2. Individual cells were manually tracked using ImageJ software. Net displacement, migration speed, and directionality were calculated using the Chemotaxis Tool plugin. [1]
- Mitosis Assay: Cells entering mitosis were manually scored based on morphology (change from flat and spread to a round, adhesive state). The percentage of cells entering mitosis per hour was calculated. [1]
- Luciferase Activity Assay: 293T cells were transfected with pGL3-HDM2-luc (HDM2-Firefly luciferase reporter) and Renilla luciferase co-reporter. Twenty-four hours post-transfection, cells were treated with DMSO or 25 µM SMIFH2 for 5 hours. Firefly luciferase activity was measured using the Dual-Luciferase Reporter Assay System and normalized against total protein content. [1]
- Knockdown Experiments: Stable mDia2 knockdown in MDA-MB-231 cells was achieved using shRNA (TRCN0000150903 and TRCN0000150850). Stable p53 knockdown in A375 and MDA-MB-231 cells was done using retroviral infection with pRS-p53. Stable p53 knockdown in U2OS cells was achieved using lentiviral shRNA (TRCN000003755 and TRCN000003756). Transient knockdowns were also performed using siRNA for mDia2 in U2OS cells. [1]

The active concentration of SMIFH2 appears to drop relatively fast inside cells, as the depolymerization-repolymerization cycles of actin and microtubules are likely due to intracellular SMIFH2 breakdown/inactivation. This was supported by an experiment where replacing the SMIFH2-containing medium every 2 hours caused a progressive and persistent depolymerization of the cytoskeleton, ruling out compound instability in the medium as the cause. [1]

High concentrations of SMIFH2 (greater than 25 µM, e.g., 50 µM) trigger rapid cell death and cytotoxic effects across all tested cell lines (U2OS, HCT116, 293T, A375, MDA-MB-231, MEFs). This event occurs independently of p53 expression. [1]

[1]. SMIFH2 has effects on Formins and p53 that perturb the cell cytoskeleton. Sci Rep. 2015 Apr 30;5:9802.

SMIFH2 is a cell-permeable small molecule inhibitor of Formin Homology 2 (FH2) domains, originally identified to inhibit Formin-dependent actin polymerization. It is used as a pharmacological Formin blocker in loss-of-function studies. [1]
- The study recommends using short incubation times (< 1 hour) and moderate concentrations (< 25 µM) of SMIFH2 to minimize confounding effects induced by loss of p53 and cytotoxicity. Under these conditions, SMIFH2 remains a useful tool to study Formins. [1]
- The study shows that the effects of SMIFH2 are not solely due to Formin inhibition, as it also downregulates p53 and p300. The cytoskeletal remodeling observed after longer treatments (>1 hour) is partly contributed by the loss of p53. [1]
- The observed depolymerization-repolymerization cycles and recovery of normal morphology after 16 hours suggest that the inhibition of Formins by SMIFH2 is transient and reversible, potentially due to intracellular drug decay and variable affinities for different Formins. [1]

C15H9N2O3SBR

377.21256

375.952

340316-62-3

2258538

Light yellow to green yellow solid powder

3.214

1

4

2

22

537

0

O=C1/C(/C(NC(=S)N1C2=CC=CC(Br)=C2)=O)=C/C3=CC=CO3

MVFJHEQDISFYIS-XYOKQWHBSA-N

InChI=1S/C15H9BrN2O3S/c16-9-3-1-4-10(7-9)18-14(20)12(13(19)17-15(18)22)8-11-5-2-6-21-11/h1-8H,(H,17,19,22)/b12-8+

(5E)-1-(3-bromophenyl)-5-(furan-2-ylmethylidene)-2-sulfanylidene-1,3-diazinane-4,6-dione

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)

DMSO : ~125 mg/mL (~331.38 mM)

Solubility in Formulation 1: 2.08 mg/mL (5.51 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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