LIMK2
T56-LIMKi (T5601640) primarily targets LIM kinase 2 (LIMK2) with high specificity, and also inhibits LIMK1 [1]
T56-LIMKi (T5601640) acts as an inhibitor of both LIMK1 and LIMK2, interfering with their kinase activity towards the substrate cofilin [2]

T56-LIMKi can cause a decrease in Panc-1 tumor size and an inhibition of cofilin phosphorylation in vivo. Tumor volume in mice given T56-LIMKi (60 mg/kg) is significantly lower than in control mice[1].

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1. In a nude mouse Panc-1 xenograft model, oral administration of T56-LIMKi (T5601640) (60 mg/kg) significantly reduced tumor volume over a 35-day treatment period (means ± S.E.M, n=8, P < 0.05); tumor weight was also lower in the treatment group compared to vehicle control [1]
2. T56-LIMKi (T5601640) decreased p-cofilin levels in Panc-1 tumor xenografts (normalized to total cofilin, means ± S.E.M, n=8, P < 0.05), confirming its target engagement in vivo [1]

T56-LIMKi (also known as T5601640) is a powerful and focused inhibitor of LIMK2 (LIM kinase 2), exhibiting an IC50 of 35.2 μM in a cell assay using Panc-1 cells.

T56-LIMKi can cause Panc-1 tumor shrinkage and cofilin phosphorylation inhibition in vivo. Tumor volume in mice treated with T56-LIMKi (60 mg/kg) is significantly lower than in control mice[1].

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1. HeLa Cell p-Cofilin Analysis: HeLa cells stably expressing LIMK1, LIMK2, or empty vector (pcDNA3) were serum-starved for 24 h, then treated with 50 μM T56-LIMKi (T5601640) for 2 h. Cell lysates were prepared, and p-cofilin, total cofilin, and β-tubulin levels were quantified by Western blotting; results were normalized to β-tubulin and expressed as a percentage of untreated control (mean ± SD, n = 3) [1]
2. Cancer Cell Proliferation Assay: Panc-1, A549, U87, and ST88-14 cells were seeded in culture plates and treated with varying concentrations of T56-LIMKi (T5601640) or 0.1% DMSO (control). After 6 days of culture, cells were directly counted to assess proliferation; data were presented as mean ± SEM (n = 9) [1]
3. NF1-Depleted MEF p-Cofilin and ROCK Inhibitor Assay: NF1-depleted MEFs were serum-starved for 24 h and treated with different concentrations of Y-27632 for 24 h, followed by 50 μM T56-LIMKi (T5601640) for 2 h in 10% FCS. Cell lysates were analyzed by Western blotting for p-cofilin, normalized to β-tubulin (mean ± SD, n = 3) [1]
4. NF1⁻/⁻ MEF Proliferation Assay: NF1⁻/⁻ MEFs were seeded and cultured with various concentrations of T56-LIMKi (T5601640) or control for 5 days. Cells were counted, and inhibition curves were generated (mean ± SEM, n = 9) [2]
5. Actin Stress Fiber Staining Assay: NF1⁻/⁻ MEFs were seeded in 6-well plates (2.5 × 10⁴ cells/well), serum-starved (0.5% FCS) and treated with T56-LIMKi (T5601640) for 24 h. Cells were fixed, permeabilized, and stained with rhodamine-phalloidin; the percentage of cells with stress fibers was counted in 100 cells per slide (mean ± SD, n = 3) [2]
6. Scratch Wound Migration Assay: Wild-type and NF1⁻/⁻ MEFs were plated (1.5 × 10⁵ cells/35-mm plate), serum-starved (0.5% FCS) and treated with 50 μM T56-LIMKi (T5601640) or control. A scratch wound was made, and gap width was measured at different time points; data were expressed as a percentage of initial gap width (mean ± SD, n = 9) [2]
7. Soft Agar Colony Formation Assay: NF1⁻/⁻ MEFs were seeded in soft agar with different concentrations of T56-LIMKi (T5601640) and cultured for 14 days. Colonies were stained and counted; results were presented as mean ± SD (n = 5) [2]

Mice: In a 0.5% carboxymethylcellulose solution, T56-LIMKi is dissolved. Panc-1 xenografts are injected into mice. Seven days later, treatment is initiated. Mice in the control group are given only the vehicle (0.5% CMC) in the gavage, while mice in the two experimental groups are given daily oral non-toxic doses of T56-LIMKi (30 or 60 mg/kg)[1].

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1. Panc-1 Xenograft Tumor Model: Panc-1 pancreatic cancer cells were subcutaneously implanted into the right flank of nude mice. After tumor establishment, mice were divided into treatment groups (n=8) and administered T56-LIMKi (T5601640) at a dose of 60 mg/kg via an unspecified route (oral administration implied) for 35 days. Tumor volumes were measured regularly, and body weights were recorded to assess toxicity. After treatment, tumors were excised, weighed, and photographed; tumor lysates were prepared for p-cofilin analysis by Western blotting [1]

1. In nude mice, T56-LIMKi (T5601640) was well tolerated at a dose of 60 mg/kg for 35 consecutive days, and no significant changes in body weight were observed during the treatment period [1].

[1]. Novel LIMK2 Inhibitor Blocks Panc-1 Tumor Growth in a mouse xenograft model. Oncoscience. 2014 Jan 1;1(1):39-48. eCollection 2014.

[2]. Computer-based identification of a novel LIMK1/2 inhibitor that synergizes with salirasib to destabilize the actin cytoskeleton. Oncotarget. 2012 Jun;3(6):629-39.

1. T56-LIMKi (T5601640) was identified using a combination of computational molecular modeling (based on the structural similarity between the active sites of EphA3 and LIMK2) and classical biochemical techniques [1][2]. 2. This inhibitor works by blocking LIMK2-mediated cofilin phosphorylation, thereby activating cofilin and subsequently remodeling the actin cytoskeleton, thereby inhibiting cancer cell proliferation, migration, and tumor growth [1]. 3. T56-LIMKi (T5601640) has a much higher specificity for LIMK2 than for LIMK1, making it a valuable tool for studying the specific function of LIMK2 in cancer and neurological diseases [1]. 4. T56-LIMKi (T5601640) When used in combination with salirasib (a Ras inhibitor), this drug has a synergistic effect on actin cytoskeleton instability and cell proliferation inhibition in NF1-deficient cells, suggesting its potential application value in the combined treatment of neurofibromatosis and cancer [2].

C19H14F3N3O3

389.33

389.098

C, 58.62; H, 3.62; F, 14.64; N, 10.79; O, 12.33

924473-59-6

9438169

White to off-white solid powder

1.4±0.1 g/cm3

403.4±45.0 °C at 760 mmHg

197.8±28.7 °C

0.0±0.9 mmHg at 25°C

1.618

2.85

2

7

4

28

572

0

O=C(C1=CC(C)=NO1)NC1C=C(C(NC2C=C(C(F)(F)F)C=CC=2)=O)C=CC=1

XVOKFRPKSAWELK-UHFFFAOYSA-N

InChI=1S/C19H14F3N3O3/c1-11-8-16(28-25-11)18(27)24-14-6-2-4-12(9-14)17(26)23-15-7-3-5-13(10-15)19(20,21)22/h2-10H,1H3,(H,23,26)(H,24,27)

3-methyl-N-[3-[[3-(trifluoromethyl)phenyl]carbamoyl]phenyl]-1,2-oxazole-5-carboxamide

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:≥ 40 mg/mL
Water:
Ethanol:

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.42 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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