RSK1/Ribosomal S6 Kinase (IC50 = 6 nM); RSK1 (IC50 = 5 nM); RSK1 (IC50 = 4 nM)
LJH685 targets the p90 ribosomal S6 kinase (RSK) family, which includes RSK1, RSK2, and RSK3. These kinases are downstream effectors of the MAPK/ERK signaling pathway and are involved in regulating cell proliferation, survival, and protein synthesis. LJH685 acts as an ATP-competitive inhibitor, binding to the ATP-binding pocket of the kinases and preventing their phosphorylation activity. It inhibits RSK1, RSK2, and RSK3 with IC50 values of 6 nM, 5 nM, and 4 nM, respectively. The compound shows high selectivity for RSKs over a panel of 96 other kinases, making it a valuable tool for studying RSK-specific functions.

LJH685 (0.01-100 μM; 72 hours) has effective EC50 values of 0.73 and 0.79 μM, respectively, for inhibiting the growth of MDA-MB-231 and H358 cells in soft agar.
LJH685 (0.1-10 μM; 4 hours) efficient at submicromolar concentrations and nearly completely inhibits YB1 phosphorylation at higher concentrations[1].
In vitro, LJH685 inhibits RSK1, RSK2, and RSK3 biochemical activities with IC50 values of 6 nM, 5 nM, and 4 nM, respectively. It is selective for RSKs over a panel of 96 other kinases. In MAPK pathway-dependent cancer cell lines, LJH685 shows antiproliferative effects, causes cell-cycle regulation, and induces apoptosis. It modulates YB1 phosphorylation by potently and selectively inhibiting RSK in cells. These in vitro activities confirm its potent and selective RSK inhibition.

The RSK inhibitor, LJH685, suppressed BLBC cell tumourigenesis in vivo by disturbing YB-1-KLF5 axis. Our data suggest that YB-1 positively regulates KLF5 at multiple levels to promote BLBC progression. The novel RSK2-YB-1-KLF5-KRT16/Ly6D axis provides candidate diagnostic markers and therapeutic targets for BLBC.[2]
To test this, we evaluated the anti-tumour effect of LJH685 in an HCC1806 orthotopic xenograft mouse model. As expected, we observed significant suppression of tumour growth following LJH685 administration (Fig. 7H, I), although this compound showed low solubility in water and a short half-life in blood. There was no marked weight loss in the LJH685-treated mice, suggesting its toxicity was acceptable (Supplementary Fig. S8E). Furthermore, we demonstrated that LJH685 decreased the expression of YB-1 pS102, KLF5, KRT16, Ly6D, and Cyclin D1, but increased cleaved-PARP1 in the xenograft tumour tissues (Supplementary Fig. S8F). Taken together, LJH685 efficiently inhibits HCC1806 cell growth in vivo and therefore represents a potential drug for the treatment of BLBC.[2]
In vivo, LJH685 modulates YB1 phosphorylation by potently and selectively inhibiting RSK. In MAPK pathway-dependent cancer cell lines, it shows antiproliferative effects, causes cell-cycle regulation, and induces apoptosis. Detailed in vivo efficacy data from animal models are not extensively reported in the available literature. As a potent and selective RSK inhibitor, it is expected to have therapeutic potential in cancers driven by MAPK pathway activation.

Recombinant full-length RSK protein is used to measure the enzymatic activity of RSK isoforms 1, 2, and 3 (PV4049, PV4051, and PV3846). RSK1 (1 nmol/L), RSK2 (0.1 nmol/L), or RSK3 (1 nmol/L) is allowed to phosphorylate 200 nmol/L peptide substrate (biotin-AGAGRSRHSSYPAGT-OH) in the presence of ATP at concentration equal to the Km for ATP for each enzyme (RSK1, 5 μmol/L; RSK2, 20 μmol/L; and RSK3, 10 μmol/L) and appropriate dilutions of RSK inhibitors.
Non-cell-based enzyme assays for LJH685 use purified RSK1, RSK2, and RSK3 kinases. The compound is incubated with the kinase, a peptide substrate, and ATP at varying concentrations. Kinase activity is measured by quantifying substrate phosphorylation using techniques such as radiometric detection (e.g., [γ-33P]-ATP incorporation), fluorescence polarization, or ELISA. IC50 values for enzyme inhibition are determined from dose-response curves. Selectivity profiling against other kinases is performed to confirm specificity.

By seeding 1000 cells per well on 96-well tissue culture-treated plates with cell growth medium, it is possible to measure cell growth under the attached conditions. After 72 hours, cell growth is evaluated by adding CellTiter Glo reagent in accordance with manufacturer's instructions. Appropriate dilutions of the compound are added to the medium above the cells.
Cellular assays for LJH685 utilize MAPK pathway-dependent cancer cell lines. Cells are treated with the compound at various concentrations for specified durations. Cell proliferation is assessed using standard viability assays (e.g., MTT, CellTiter-Glo). Cell cycle analysis is performed by flow cytometry after propidium iodide staining. Apoptosis is evaluated using Annexin V/PI staining or caspase activity assays. YB1 phosphorylation is measured by Western blotting using phospho-specific antibodies.

Administration of LJH685 in tumour burden mice[2]
HCC1806 cells (6 × 105) were injected subcutaneously into both the left and right mammary fat pads of twelve female nude mice (6–7-week-old). After seven days, the tumour volume and weight of mice were measured, and mice were randomly distributed into two groups. The mice were then treated with LJH685 or NC by intraperitoneal injection daily. The tumour volume and mouse weight were measured every other day. The mice were sacrificed on day 21, and the tumours were harvested and weighed. LJH685 was prepared by adding each of the following solvents in sequential order; 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline. The drug solution was freshly prepared to avoid freeze thawing that could cause drug precipitation.
In vivo animal models for LJH685 would include xenograft studies in immunodeficient mice bearing MAPK pathway-dependent cancer cell lines. The compound would be administered orally or via other routes at various doses. Tumor growth inhibition would be monitored over time. Pharmacodynamic markers such as YB1 phosphorylation and RSK target gene expression in tumor tissues would be assessed. Detailed protocols are not extensively reported in the available literature.

Pharmacokinetic properties of LJH685 include a molecular weight of 381.42 g/mol and molecular formula C22H21F2N3O. CAS number is 1627710-50-2. Purity is >98%. The compound is soluble in DMSO at 66 mg/mL (173.03 mM) and in ethanol at 17 mg/mL (44.57 mM), but insoluble in water. Storage conditions: powder at -20°C for 3 years; in solution at -20°C for 6 months. Detailed PK parameters are not extensively reported.

Detailed toxicological data for LJH685 are not extensively reported in the available literature. As a research compound, its safety profile would need to be established through standard preclinical toxicity assessments. The compound is supplied for research use only. Given its mechanism of RSK inhibition, potential effects on cell proliferation and survival in normal tissues would be a key safety consideration.

[1]. Mol Cancer Res. 2014 May;12(5):803-12.

[2]. YB-1 is a positive regulator of KLF5 transcription factor in basal-like breast cancer. Cell Death & Differentiation volume 29, pages1283–1295 (2022)

The p90 ribosomal S6 kinase (RSK) family is a class of serine/threonine kinases expressed in various cancers, and their substrate phosphorylation is closely related to the direct regulation of cell survival, proliferation, and cell polarity. This study characterized and introduced LJH685 and LJI308, the most selective and potent RSK inhibitors known to date. Structural analysis confirmed that LJH685 binds to the ATP-binding site of the RSK2 N-terminal kinase and revealed that this inhibitor exhibits an unusual non-planar conformation, explaining its excellent selectivity for RSK family kinases. LJH685 and LJI308 effectively inhibited RSK activity both in vitro and in cells. Furthermore, RSK cellular inhibition and its inhibition of YB1 Ser102 phosphorylation are closely associated with cell growth inhibition, but this correlation was only observed under non-anchor-dependent growth conditions and in only a subset of the tested cell lines. Therefore, RSK inhibition reveals dynamic functional responses among inhibitor-sensitive cell lines, highlighting the heterogeneity of RSK-dependent inhibition in cancer. [1] Y-box binding protein 1 (YB-1) is a well-known oncogene that is highly expressed in a variety of cancers, including basal-like breast cancer (BLBC). In addition to being a transcription factor, YB-1 has recently been defined as an epigenetic regulator involving RNA 5-methylcytosine. However, its specific targets and oncogenic functions remain unclear. Here, based on clinical databases, we demonstrated that in breast cancer patients, the expression of Kruppel-like factor 5 (KLF5) and YB-1 was positively correlated, while it was negatively correlated with the expression of Dachshund homolog 1 (DACH1). Mechanistically, YB-1 not only enhances KLF5 expression through transcriptional activation that can be inhibited by DACH1, but also stabilizes KLF5 mRNA in a manner dependent on RNA 5-methylcytosine modification. In addition, ribosomal S6 kinase 2 (RSK2)-mediated phosphorylation of YB-1 at Ser102 promotes the formation of the YB-1/KLF5 transcriptional complex, which co-regulates the expression of BLBC-specific genes keratin 16 (KRT16) and lymphocyte antigen 6 family member D (Ly6D), thereby promoting cancer cell proliferation. The RSK inhibitor LJH685 inhibits tumorigenesis of BLBC cells in vivo by interfering with the YB-1-KLF5 axis. Our data suggest that YB-1 positively regulates KLF5 at multiple levels to promote BLBC progression. The newly discovered RSK2-YB-1-KLF5-KRT16/Ly6D axis provides potential diagnostic biomarkers and therapeutic targets for BLBC. [2]
LJH685 is also known as LJH 685 and LJH-685. It has CAS number 1627710-50-2. It is a potent, specific, and selective RSK inhibitor that inhibits RSK1, RSK2, and RSK3 with IC50 values of 6 nM, 5 nM, and 4 nM, respectively. It is selective for RSKs over a panel of 96 other kinases. It modulates YB1 phosphorylation in cells and shows antiproliferative effects, cell-cycle regulation, and apoptosis induction in MAPK pathway-dependent cancer cell lines. Purity is >98%.

C22H21F2N3O

381.42

381.165

C, 69.28; H, 5.55; F, 9.96; N, 11.02; O, 4.19

1627710-50-2

1627710-50-2

73010393

White to off-white solid powder

1.3±0.1 g/cm3

481.1±45.0 °C at 760 mmHg

244.7±28.7 °C

0.0±1.3 mmHg at 25°C

1.601

3.31

1

6

3

28

482

0

C1=C(C2C(C3C=CC(N4CCN(CC4)C)=CC=3)=CC=NC=2)C=C(C(O)=C1F)F

IKUFKDGKRLMXEX-UHFFFAOYSA-N

InChI=1S/C22H21F2N3O/c1-26-8-10-27(11-9-26)17-4-2-15(3-5-17)18-6-7-25-14-19(18)16-12-20(23)22(28)21(24)13-16/h2-7,12-14,28H,8-11H2,1H3

2,6-difluoro-4-[4-[4-(4-methylpiperazin-1-yl)phenyl]pyridin-3-yl]phenol

LJH685; LJH685; LJH685; NVP LJH685; NVP-LJH685;NVPLJH685; NVP LJH 685; 2,6-difluoro-4-[4-[4-(4-methylpiperazin-1-yl)phenyl]pyridin-3-yl]phenol; 2,6-Difluoro-4-{4-[4-(4-Methylpiperazin-1-Yl)phenyl]pyridin-3-Yl}phenol; CHEMBL3604793; 27CZQ807C1; NVP-LJH-685

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: ~20 mg/mL (~52.4 mM)
Ethanol: ~10mg/mL (~26.2 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (2.62 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1 mg/mL (2.62 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 10.0 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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Solubility in Formulation 3: ≥ 1 mg/mL (2.62 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 10.0 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.)