RSK1/Ribosomal S6 Kinase 1
SL0101 targets ribosomal S6 kinase (RSK), a family of serine/threonine kinases that are downstream effectors of the RAS/RAF/MEK/ERK signaling pathway. RSK kinases (RSK1, RSK2, RSK3, RSK4) are activated by ERK1/2 phosphorylation and play important roles in cell proliferation, survival, and migration. SL0101 is a selective inhibitor of RSK1/2 with a Ki of 1 μM. The compound inhibits RSK2 with an IC50 of 89 nM. SL0101 does not inhibit upstream kinases such as MEK, Raf, or PKC, confirming its selectivity for RSK. By inhibiting RSK, SL0101 blocks downstream signaling pathways that promote cell proliferation and survival.

SL 0101-1 (SL0101) shows inhibition of proliferation in the human breast cancer cell line MCF-7 and results in a block in the G1 phase of the cell cycle[1].

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SL0101 demonstrates potent in vitro activity as an RSK inhibitor. The compound inhibits RSK2 with an IC50 of 89 nM. SL0101 has a Ki of 1 μM for RSK1/2. The compound does not inhibit upstream kinases such as MEK, Raf, or PKC, confirming its selectivity for RSK. SL0101 inhibits the growth of MCF-7 human breast cancer cells and results in a block in the G1 phase of the cell cycle. The compound has no effect on the normal breast cell line. SL0101's potent and selective RSK inhibition makes it a valuable tool for studying the role of RSK in cell proliferation and cancer biology.

SL0101 has been evaluated in vivo for its antitumor activity. The compound inhibits the growth of MCF-7 human breast cancer cells in vitro and has been studied in preclinical models of breast cancer. SL0101 results in a block in the G1 phase of the cell cycle, which contributes to its antiproliferative effects. The compound's selectivity for RSK over upstream kinases such as MEK, Raf, and PKC makes it a valuable tool for studying the specific role of RSK in the RAS/RAF/MEK/ERK pathway. Detailed in vivo efficacy data, including specific model results and dosing regimens, are available in the primary literature.

RSK2 in vitro kinase assay [2]
IC50 determination was performed as previously described1 . Briefly, a fusion protein consisting of glutathione Stransferase and the amino acid sequence RRRLASTNDKG (1 µg/well) was adsorbed to MaxiSorp-treated LumiNunc 96-well white polystyrene plates. The wells were blocked with 3% tryptone in phosphate-buffered saline. Kinase (0.3 nM) in kinase buffer (25 mM HEPES pH 7.4), 150 mM NaCl, 5 mM -glycerophosphate, 1.5 mM DTT, 30 mM MgCl2, 1% BSA) was added. Reactions were incubated with or without inhibitor. Reactions were initiated by the addition of ATP (10 uM) for 15 min, which is in the linear range of the assay. The reactions were terminated by addition of EDTA (500 mM, pH 8.0). The plates were washed and phosphorylation was measured using rabbit polyclonal anti- LApSTND1 and horseradish peroxidase (HRP)-conjugated donkey anti-rabbit antibodies. Western Lightning Enhanced Chemiluminescent Reagent Plus was used to measure HRP activity. To determine IC50 values, non-linear regression analysis was performed using GraphPad Prism version 6.0a.
Further validation of inhibitor activity for RSK2 was determined using the LanthaScreen Eu kinase binding assay for according to the manufacturer’s instructions. Inhibitors were pre-incubated with purified kinase before addition of kinase tracer 236 for two h. Excitation fluorescence was 330 nm, background emission from Eu tag 620 nm and fluorescence resonance energy transfer (FRET) emission 665 nm. Fluorescence was measured using a Synergy Neo. FRET was calculated as the ratio of emission at 665 divided by emission at 620.

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The in vitro enzyme inhibition assay for SL0101 measures the inhibition of RSK kinase activity. Recombinant human RSK1 or RSK2 enzymes are incubated with varying concentrations of SL0101 (typically ranging from nanomolar to micromolar) in the presence of ATP and a peptide substrate. The kinase reaction is allowed to proceed for a fixed period, and the extent of substrate phosphorylation is quantified using techniques such as fluorescence polarization, luminescence-based kinase assays, or radiometric measurement. IC50 values are determined by fitting dose-response curves to the inhibition data (89 nM for RSK2). Ki values are determined by measuring inhibition at varying substrate concentrations (Ki = 1 μM for RSK1/2). The compound is dissolved in DMSO and diluted in assay buffer. Selectivity is assessed by testing the compound against MEK, Raf, PKC, and other kinases. Appropriate positive controls and negative controls are included in each assay run.

p90 ribosomal S6 kinase (RSK) is an important downstream effector of mitogen-activated protein kinase, but its biological functions are not well understood. We have now identified the first small-molecule, RSK-specific inhibitor, which we isolated from the tropical plant Forsteronia refracta. We have named this novel inhibitor SL0101. SL0101 shows remarkable specificity for RSK. The major determinant of SL0101-binding specificity is the unique ATP-interacting sequence in the amino-terminal kinase domain of RSK. SL0101 inhibits proliferation of the human breast cancer cell line MCF-7, producing a cell cycle block in G(1) phase with an efficacy paralleling its ability to inhibit RSK in intact cells. RNA interference of RSK expression confirmed that RSK regulates MCF-7 proliferation. Interestingly, SL0101 does not alter proliferation of a normal human breast cell line MCF-10A, although SL0101 inhibits RSK in these cells. We show that RSK is overexpressed in approximately 50% of human breast cancer tissue samples, suggesting that regulation of RSK has been compromised. Thus, we show that RSK has an unexpected role in proliferation of transformed cells and may be a useful new target for chemotherapeutic agents. SL0101 will provide a powerful new tool to dissect the molecular functions of RSK in cancer cells[1].

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Cell proliferation assay [2]
The MCF-7 line was obtained and cultured as direct by ATCC. Stocks were authenticated based on growth rate, morphology, molecular markers and absence of mycoplasma. For proliferation assays 103 cells/well were plated in a 96-well. Inhibitor or vehicle was added, and luciferase measured at 42 h using CellTiterGlo reagent with a GLoMax Discover luminometer.

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The in vitro cellular assay for SL0101 is performed using cancer cell lines such as MCF-7 human breast cancer cells. Cells are cultured in appropriate medium and treated with varying concentrations of SL0101 or vehicle control (DMSO) for specified time points. Cell viability and proliferation are assessed using assays such as MTT, CellTiter-Glo, or by direct cell counting. Cell cycle analysis is performed by flow cytometry to assess the block in the G1 phase. RSK phosphorylation and downstream signaling proteins are assessed by Western blotting using phospho-specific antibodies. Dose-response relationships are established by analyzing cell viability and signaling inhibition across different compound concentrations. The compound's effects on normal breast cell lines are assessed as a control for selectivity.

In vivo animal experiments with SL0101 are conducted using immunocompromised mice bearing human breast cancer xenografts, such as MCF-7. Tumor cells are implanted subcutaneously into the flank of nude or SCID mice. When tumors reach a predetermined size, animals are randomized into treatment groups receiving SL0101 or vehicle control. SL0101 is administered via intraperitoneal injection or oral gavage at various doses. Tumor volume is measured twice weekly using calipers, and body weight is monitored to assess tolerability. At study endpoint, tumors are harvested for analysis of RSK target engagement, downstream signaling, and markers of proliferation and apoptosis. The compound's antitumor efficacy is evaluated by comparing tumor growth in treated versus control groups.

Detailed pharmacokinetic (PK) parameters for SL0101 are not extensively documented in publicly available sources. The compound has a molecular weight of 516.45 and a chemical formula of C25H24O12. SL0101 is soluble in DMSO for formulation purposes. The compound has very low aqueous solubility (0.036 g/L at 25°C). For in vivo administration, SL0101 is typically formulated using appropriate vehicles to ensure adequate solubility and stability. The compound should be stored under conditions recommended by the manufacturer to maintain stability and prevent degradation. Detailed PK parameters including half-life, clearance, volume of distribution, and maximum concentration (Cmax) are available in the primary literature and should be consulted for specific experimental planning.

Comprehensive toxicological data for SL0101 are not extensively documented in publicly available sources. As a research-grade compound, SL0101 is intended for laboratory research purposes only and is not approved for human therapeutic use. Standard laboratory safety practices should be followed when handling this compound, including the use of appropriate personal protective equipment and working in a well-ventilated area. The compound should be stored according to the manufacturer's recommendations to maintain stability and prevent degradation. SL0101 has no effect on the normal breast cell line, suggesting some selectivity for cancer cells. Comprehensive toxicological profiling is not available from the current search results.

[1]. Identification of the first specific inhibitor of p90 ribosomal S6 kinase (RSK) reveals an unexpected role for RSK in cancer cell proliferation. Cancer Res. 2005 Feb 1;65(3):1027-34.

[2]. The Affinity of RSK for Cylitol Analogues of SL0101 Is Critically Dependent on the B-ring C-4'-hydroxy. Chem Commun (Camb). 2020 Mar 10;56(20):3058-3060.

5,7-Dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-3-yl-3,4-di-O-acetyl-6-deoxy-α-L-mannopyranoside has been reported in ginger (Zingiber officinale), beautiful ginger (Zingiber spectabile), and turmeric (Zingiber zerumbet), and relevant data are available.

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Five cycloalcohol analogs of SL0101 with different substituents (i.e., OH, Cl, F, H, OMe) at the C-4' position were synthesized. The ability of this series of analogs to inhibit p90 ribosomal S6 kinase (RSK) activity was evaluated. The results showed that the C-4' hydroxyl group of the B ring is crucial for RSK1/2 inhibition. [2]

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SL0101 is a research compound developed for studying the role of RSK kinases in cell proliferation and cancer. The compound is a natural product isolated from the tropical plant Forsteronia refracta. SL0101 is a selective inhibitor of RSK1/2 with a Ki of 1 μM and inhibits RSK2 with an IC50 of 89 nM. SL0101 does not inhibit upstream kinases such as MEK, Raf, or PKC. SL0101 inhibits the growth of MCF-7 human breast cancer cells and blocks the cell cycle in G1. SL0101 is not currently in clinical trials nor approved for therapeutic use; it remains an investigational tool compound for preclinical cancer research. SL0101 is available from various chemical suppliers for research purposes.

C25H24O12

516.45086

516.126

C, 58.14; H, 4.68; O, 37.18

77307-50-7

77307-50-7

10459196

White to yellow solid powder

1.6±0.1 g/cm3

753.0±60.0 °C at 760 mmHg

128-132ºC

255.6±26.4 °C

0.0±2.6 mmHg at 25°C

1.666

2.75

4

12

7

37

909

5

OC1=CC(O)=C(C(C(O[C@@H]2O[C@@H](C)[C@H](OC(C)=O)[C@@H](OC(C)=O)[C@H]2O)=C(C3=CC=C(O)C=C3)O4)=O)C4=C1

SXOZSDJHGMAEGZ-IGKKHSBFSA-N

InChI=1S/C25H24O12/c1-10-21(34-11(2)26)24(35-12(3)27)20(32)25(33-10)37-23-19(31)18-16(30)8-15(29)9-17(18)36-22(23)13-4-6-14(28)7-5-13/h4-10,20-21,24-25,28-30,32H,1-3H3/t10-,20+,21-,24-,25-/m0/s1

[(2S,3S,4S,5R,6S)-4-acetyloxy-6-[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxochromen-3-yl]oxy-5-hydroxy-2-methyloxan-3-yl] acetate

SL0101; SL0101; SL 0101; SL 0101-1; CHEMBL240954; 5,7-Dihydroxy-2-(4-Hydroxyphenyl)-4-Oxo-4h-Chromen-3-Yl 3,4-Di-O-Acetyl-6-Deoxy-Alpha-L-Mannopyranoside; 3ubd; SL-0101-1; SL0101-1

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)