TH-257 A chemical probe for LIMK1/2

Probe and control are available from Cayman.

Probe is available from Sigma.


LIM kinases belong to the family of cytoplasmic tyrosine-like kinases with dual specificity (serine/threonine and tyrosine). However, known LIMK substrate are usually phosphorylated at serine and threonine residues LIM kinases comprises LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2) which show 50% sequence identity in human. Both LIMK1 and LIMK2 present with a unique domain organization containing two N-terminal LIM domains, a PDZ domain, a proline/serine-rich domain and a C-terminal kinase domain [1].

Both proteins are expressed widely in embryonic and adult tissues, but show some cell-type specific expression. Accordingly, the two kinases have overlapping functions, but appear non-redundant. Knockout studies in mice show that LIMK1 is required for development of the central nervous system [2], whereas LIMK2 knockout impairs the activity of testicular germ cells [3].

LIMKs are effectors of cell morphology and motility and apoptosis by regulating the actin cytoskeleton. The LIMKs signal downstream from Rho GTPases and are activated by phosphorylation of the activation loop by upstream kinases, including Rho kinase (ROCK), PAK1/2/4 and MRCKα. The best characterized LIMK substrates are cofilin1 (non-muscle cofilin), cofilin2 (muscle cofilin) and destrin (actin depolymerizing factor, ADF). Phosphorylation of cofilin serine-3 inactivates the actin severing ability promoting F-actin polymerization, stress fibre formation and focal adhesion formation [4].

LIM kinases can shuttle between the cytoplasm and the nuclear compartment of a cell, a process tightly regulated by association with other partners such as p57kip2 and phosphorylation in the activation segment by PAK kinases [1]. Inhibition of LIMK hyper-stabilizes mitotic spindles inducing a G2/M cell cycle block suggesting an important role for these kinases in microtubule dynamics [5].

Increased phosphorylation of LIMK1 has been reported in neurons in areas affected with Alzheimer Disease [6]. LIM kinases play important roles in cancer metastasis like highly invasive prostate and breast cancer, which is reversed by gene silencing [7, 8]. LIMK1 overexpression is also found in malignant melanoma, as well as most tumour cell lines. Other applications for LIMK inhibitors are open-angle glaucoma [9]. In addition, LIMK1 interacts with the long isoform of the type II bonemorphogenetic protein (BMP) receptor contributing to the pathology of Fragile X syndrome, a common inherited form of intellectual disability [10].

TH-257 is a chemical probe for LIMK1 and LIMK2. TH-257 is an allosteric inhibitor targeting a binding pocket induced by an αC and DFG-out conformation. It potently inhibits cofilin phosphorylation with an IC50 of 84 nM for LIMK1 and 39 nM for LIMK2 in a RapidFire MS assay. TH257 is exquisitely selective and no significant activity against the wider kinome has been observed in the KINOMEscan assay (Dx) at 1 μM (IC50 >> 50 % inhibition). In a life cell NanoBRET assay (Promega) TH257 has an IC50 of 250 nM against ectopically expressed full-length LIMK1 and 150 nM LIMK2, respectively.

A chemically related negative control compound, TH-263, is provided.

Probe Negative Control
LIJTF500025 LIJTF500120

Takeda in collaboration with the SGC has developed LIJTF500025, a potent and selective inhibitor for the LIMK1/2. LIJTF500025 binding to LIMK1/2 was confirmed by DSF and ITC. Its crystall structure revealed an allosteric binding mode (Type III) that explains he high selectivity within the humane kinome. The cellular activity was determined by NanoBRET and revealed EC50 values of 82 nM on LIMK1 and 52 nM on LIMK2 was just one additional off-target (RIPK1 EC50 = 6 nM). The chemical probe (LIJTF500025) is accompanied by a negative control (LIJTF500120) that is structurally closely related to the probe molecule.

Potency Against Target Family

LIJTF500025 had an KD Value of 37 nM on LIMK1 determined by ITC.


LIJTF500025 was selective in an in-house DSF kinase panel against 107 kinases. In addition, the selectivity was confirmed in the ScanMAX Kinase Panel from Eurofins (DiscoverY) against 468 kinases at a screening concentration of 1 µM.


Based on the potency and the selectivity of the chemical probe and to minimize the risk of unspecific cytotoxicity, we recommend a concentration of no higher than 1 µM for cell-based assays.

Cellular Activity

LIJTF500025 displayed an EC50 of 82 nM, 52 nM and 6.3 nM on LIMK1, LIMK2 and RIPK1 respectively in intact cells in the NanoBRET assay.

Physical and chemical properties LIJTF500025
Molecular weight479.92
Molecular formulaC24H22ClN5O4
IUPAC name(S)-2-benzyl-6-(8-chloro-5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-7-oxo-4,5,6,7-tetrahydro-2H-pyrazolo[3,4-c]pyridine-3-carboxamide
No. of chiral centres1
No. of rotatable bonds4
No. of hydrogen bond acceptors9
No. of hydrogen bond donors2
Storager. t.


SMILES: CN1C([C@H](COc2c1ccc(Cl)c2)N3CCc4c(C(N)=O)n(Cc5ccccc5)nc4C3=O)=O




Physical and chemical properties LIJTF500120
Molecular weight445,48
Molecular formulaC24H23N5O4
IUPAC name(S)-1-benzyl-6-(5-methyl-4-oxo-2,3,4,5-tetrahydrobenzo[b][1,4]oxazepin-3-yl)-7-oxo-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide
No. of chiral centres1
No. of rotatable bonds4
No. of hydrogen bond acceptors9
No. of hydrogen bond donors2
Storager. t.


InChI: InChI=1S/C24H23N5O4/c1-27-17-9-5-6-10-19(17)33-14-18(23(27)31)28-12-11-16-20(22(25)30)26-29(21(16)24(28)32)13-15-7-3-2-4-8-15/h2-10,18H,11-14H2,1H3,(H2,25,30)/t18-/m0/s1


selectivity profile

Kinome-wide selectivity profile of LIJTF500025 was determined in our in-house kinase DSF-panel comprising 107 kinases and at Eurofins (DiscoverY) at 1 µM comprising 368 kinases.

DSF-Panel against 107 kinases:

Eurofins DiscoverY (468 kinases) @ 1µM:

The negative control LIJTF500120 showed no stabilization against 107 kinases screened in our in-house DSF-Panel.

DSF-Panel against 107 kinases:

in vitro potency
cell based assay data

LIJTF500025 displayed EC50 values of 82 nM on LIMK1; 52 nM on LIMK2; and 6.3 nM on RIPK1, determined by NanoBRETTM assay.

The negative control compound LIJTF500120 displayed a EC50 value of > 50 µM on LIMK1/2 and 3.5 µM on RIPK1, determined by NanoBRETTM assay.

LIJTF500025 showed a dose dependent inhibition of the phosphorylation of cofilin in LN229 cells:

  1. Manetti F. LIM kinases are attractive targets with many macromolecular partners and only a few small molecule regulators. Med Res Rev 2012;32(5):968-998.
  2. Meng Y, Zhang Y, Tregoubov V, Janus C, Cruz L, Jackson M, Lu WY, MacDonald JF, Wang JY, Falls DL et al. Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice. Neuron 2002;35(1):121-133.
  3. Takahashi H, Koshimizu U, Miyazaki J, Nakamura T. Impaired spermatogenic ability of testicular germ cells in mice deficient in the LIM-kinase 2 gene. Dev Biol 2002;241(2):259-272.
  4. Bernard O. Lim kinases, regulators of actin dynamics. Int J Biochem Cell Biol 2007;39(6):1071-1076.
  5. Oku Y, Tareyanagi C, Takaya S, Osaka S, Ujiie H, Yoshida K, Nishiya N, Uehara Y. Multimodal effects of small molecule ROCK and LIMK inhibitors on mitosis, and their implication as anti-leukemia agents. PLoS One 2014;9(3):e92402.
  6. Heredia L, Helguera P, de Olmos S, Kedikian G, Sola Vigo F, LaFerla F, Staufenbiel M, de Olmos J, Busciglio J, Caceres A et al. Phosphorylation of actin-depolymerizing factor/cofilin by LIM-kinase mediates amyloid beta-induced degeneration: a potential mechanism of neuronal dystrophy in Alzheimer's disease. J Neurosci 2006;26(24):6533-6542.
  7. Yoshioka K, Foletta V, Bernard O, Itoh K. A role for LIM kinase in cancer invasion. Proc Natl Acad Sci U S A 2003;100(12):7247-7252.
  8. Davila M, Frost AR, Grizzle WE, Chakrabarti R. LIM kinase 1 is essential for the invasive growth of prostate epithelial cells: implications in prostate cancer. J Biol Chem 2003;278(38):36868-36875.
  9. Harrison BA, Almstead ZY, Burgoon H, Gardyan M, Goodwin NC, Healy J, Liu Y, Mabon R, Marinelli B, Samala L et al. Discovery and Development of LX7101, a Dual LIM-Kinase and ROCK Inhibitor for the Treatment of Glaucoma. ACS Med Chem Lett 2015;6(1):84-88.
  10. Kashima R, Roy S, Ascano M, Martinez-Cerdeno V, Ariza-Torres J, Kim S, Louie J, Lu Y, Leyton P, Bloch KD et al. Augmented noncanonical BMP type II receptor signaling mediates the synaptic abnormality of fragile X syndrome. Sci Signal 2016;9(431):ra58.
pk properties
co-crystal structures
synthetic schemes
materials and methods