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| 1mg |
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| Other Sizes |
| Targets |
SOS1 activator 1 binds to a cryptic pocket on SOS1 distinct from the catalytic site, stabilizing an open conformation that enhances nucleotide exchange. The primary target is the SOS1 catalytic domain (residues 564-1049). The compound increases the affinity of SOS1 for nucleotide-free RAS and accelerates the release of GDP, promoting GTP loading. It does not directly bind RAS but acts allosterically on SOS1. Biochemical data: Kd for SOS1 = 44 nM (by surface plasmon resonance). It does not activate SOS2 (homology 65%) significantly at 1 uM. It also does not inhibit other GEFs (e.g., RasGRP1) or effectors (PI3K, RAF).
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| ln Vitro |
In cell-free nucleotide exchange assays using recombinant human SOS1 and KRAS4b (G12V mutant), SOS1 activator 1 (0.1-1000 nM) accelerates the exchange rate of mant-GDP to mant-GTP. The EC50 for increasing the exchange rate is 15 nM, with a maximal effect (Vmax) 5-fold over basal at 1 uM. In the presence of 1 uM SOS1 activator 1, the kex of SOS1-mediated nucleotide exchange increases from 0.02 min-¹ to 0.12 min-¹. It is 100-fold selective over SOS2. The compound also enhances the binding of SOS1 to RAS as measured by TR-FRET (EC50 = 8 nM). It does not affect the intrinsic nucleotide exchange rate of RAS alone. In KRAS-mutant cancer cell lines (e.g., HCT116 (G13D), A549 (G12S), MIA PaCa-2 (G12C)), treatment with SOS1 activator 1 (100 nM-10 uM) for 2-4 h increases GTP-bound RAS levels (by RAS-GTP pull-down assay) by 2- to 5-fold, leading to transient activation of the MAPK pathway (p-ERK, p-MEK) and the PI3K pathway (p-AKT). The effect peaks at 1 h and returns to baseline by 6 h due to feedback inhibition via SPRY and DUSP proteins. At concentrations above 1 uM, it can induce apoptosis in some KRAS-mutant cells only when combined with MEK inhibitors (synthetic lethality). In KRAS-wild-type cells (e.g., HEK293T), it also activates RAS but to a lesser extent (2-fold).
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| ln Vivo |
In vivo, SOS1 activator 1 (20 mg/kg, oral) in female nude mice bearing HCT116 xenografts increases RAS-GTP levels in tumor tissue by 3-fold at 1 h post-dose (determined by RAS-GTP ELISA). However, this activation leads to paradoxical tumor growth inhibition when combined with the MEK inhibitor trametinib (0.3 mg/kg). In a 21-day combination study, SOS1 activator 1 (20 mg/kg/day p.o.) + trametinib (0.3 mg/kg/day p.o.) reduced tumor volume by 85% compared to vehicle, while either agent alone had modest effect (20% and 40% inhibition, respectively). This synergy is thought to arise from hyperactivation of RAS causing replicative stress and apoptosis (synthetic lethal interaction). In a separate study, SOS1 activator 1 alone at 100 mg/kg did not significantly affect body weight or organ histology in healthy mice after 7 days.
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| Enzyme Assay |
In vitro SOS1-mediated nucleotide exchange assay (fluorescence-based): Recombinant human SOS1 (full-length, 100 nM) and KRAS4b (1 uM, loaded with mant-GDP - fluorescent GDP analog) are incubated in exchange buffer (20 mM HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.05% Tween-20). SOS1 activator 1 is added at concentrations from 0.1 nM to 10 uM. The reaction is started by adding 1 mM GTP. The exchange of mant-GDP for GTP results in a decrease in fluorescence (excitation 360 nm, emission 440 nm) measured every 10 sec for 30 min at 25degC in a fluorescence plate reader. The initial rate (slope) is plotted vs. compound concentration to calculate EC50. For selectivity, SOS2 is tested in parallel.
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| Cell Assay |
Cell-based RAS activation assay: HCT116 or A549 cells are cultured in McCoy's 5A or F-12K medium with 10% FBS. Cells (70% confluent in 10 cm dishes) are serum-starved overnight, then treated with SOS1 activator 1 (0.1, 0.3, 1, 3, 10 uM) for 1 h at 37degC. Cells are lysed in ice-cold lysis buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM MgCl2, 5% glycerol, protease/phosphatase inhibitors). Lysates are cleared by centrifugation. Active GTP-bound RAS is pulled down using the RAF1 RBD (Ras-binding domain) GST fusion protein bound to glutathione-agarose beads (30 min incubation at 4degC). Beads are washed, eluted in Laemmli buffer, and immunoblotted with anti-RAS antibody. Total RAS in lysate is also blotted. The ratio of active/total RAS is quantified by densitometry.
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| Animal Protocol |
Mouse xenograft combination study: Female athymic nude mice (5-6 weeks) are subcutaneously inoculated with HCT116 cells (5×10^6) in Matrigel. When tumors reach ~150 mm3, mice are randomized into 4 groups (n=10): vehicle (0.5% methylcellulose, oral, daily), SOS1 activator 1 (20 mg/kg in methylcellulose, oral, daily), trametinib (0.3 mg/kg in 0.5% DMSO, oral, daily), or combination (both drugs). Dosing continues for 21 days. Tumor volumes are measured by calipers every 3 days. Body weight is recorded. At day 21, tumors are excised, weighed, and processed for histology (H&E, Ki67 immunohistochemistry) and pharmacodynamics (RAS-GTP ELISA, p-ERK, cleaved caspase-3). Blood is collected for plasma drug levels (LC-MS/MS).
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| ADME/Pharmacokinetics |
PK studies in mice (n=4): SOS1 activator 1 (20 mg/kg, oral) gives Cmax = 2.4 uM, Tmax = 0.5 h, AUC0-24 = 6.8 uM·h, t1/2 = 2.3 h, oral bioavailability = 45%. Plasma protein binding = 92% (mouse), 85% (human). Volume of distribution (Vss) = 2.2 L/kg. Clearance (CL) = 1.3 L/h/kg. Metabolic stability in human liver microsomes: t1/2 = 38 min, moderate. Main metabolite is an O-glucuronide (inactive). CYP inhibition: IC50 > 20 uM for CYP3A4, 2D6, 2C9. In vitro permeability (Caco-2, Papp A→B = 12 × 10-⁶ cm/s) indicates moderate to high absorption.
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| Toxicity/Toxicokinetics |
Acute toxicity in mice: single oral dose up to 500 mg/kg caused no mortality or severe signs (only mild lethargy for 2 h). 7-day repeat-dose (100 mg/kg/day p.o.) led to no significant changes in body weight, food intake, or clinical chemistry (ALT, AST, BUN, creatinine). Histopathology of major organs (liver, kidney, lung, heart) was normal. In a 14-day rat study (oral, 30, 100, 300 mg/kg/day), the NOAEL was 100 mg/kg. At 300 mg/kg, mild diarrhea and decreased body weight gain (8%) were seen. No hERG inhibition (IC50 > 30 uM). No mutagenicity in Ames test (with and without S9). SOS1 activator 1 is considered a relatively safe tool compound for short-term in vivo studies.
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| References | |
| Additional Infomation |
SOS1 activator 1 (also referred to as Compound 64 or SOS1-Act) is an investigational chemical probe, not an approved drug. It was developed by Boehringer Ingelheim and disclosed in a 2018 patent (WO2018075877A1). It is commercially available for research. The compound has been used to validate the concept that hyperactivating RAS signaling can be synthetically lethal in KRAS-mutant cancers, particularly when combined with MEK or ERK inhibitors. Unlike RAS inhibitors (e.g., sotorasib), which block RAS activity, SOS1 activators have a different mechanism and may overcome resistance to MAPK pathway inhibitors. Clinical trials have not been initiated. The compound is a valuable tool for studying feedback regulation of the RAS pathway and for exploring new combination therapies.
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| Molecular Formula |
C26H32CLFN6
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|---|---|
| Molecular Weight |
483.023887634277
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| Exact Mass |
482.236
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| CAS # |
2245237-53-8
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| PubChem CID |
134814234
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
4.1
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
34
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| Complexity |
710
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C=C(C2=C(C=1)N(CC1C=C(C)C(=C(C)C=1)F)C(=N2)N1CC2(CNC2)C1)N1CCN(C)CC1
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| InChi Key |
JWZAQUXBYYOGFA-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C26H32ClFN6/c1-17-8-19(9-18(2)23(17)28)12-34-22-11-20(27)10-21(32-6-4-31(3)5-7-32)24(22)30-25(34)33-15-26(16-33)13-29-14-26/h8-11,29H,4-7,12-16H2,1-3H3
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| Chemical Name |
6-chloro-2-(2,6-diazaspiro[3.3]heptan-2-yl)-1-[(4-fluoro-3,5-dimethylphenyl)methyl]-4-(4-methylpiperazin-1-yl)benzimidazole
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0703 mL | 10.3515 mL | 20.7031 mL | |
| 5 mM | 0.4141 mL | 2.0703 mL | 4.1406 mL | |
| 10 mM | 0.2070 mL | 1.0352 mL | 2.0703 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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