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| Targets |
The primary target of NR1 is the small G-protein Rheb (Ras homolog enriched in brain), a critical upstream activator of mTORC1. Rheb functions as a molecular switch that, in its GTP-bound active state, directly binds to and stimulates the kinase activity of mTORC1. NR1 binds directly to the switch II domain of Rheb with an IC50 of 2.1 μM in the Rheb-IVK assay, preventing the conformational changes required for Rheb-mediated mTORC1 activation. This interaction selectively inhibits the activation of mTORC1 without affecting mTORC2, which is regulated by distinct upstream mechanisms. In cellular models, NR1 suppresses the phosphorylation of the mTORC1 substrate T389pS6K1, a key downstream effector of mTORC1 that regulates protein synthesis and cell growth. Simultaneously, NR1 enhances the phosphorylation of S473pAKT in a dose-dependent manner. This paradoxical increase in AKT phosphorylation is a well-documented consequence of mTORC1 inhibition, as mTORC1 normally exerts negative feedback on AKT signaling through S6K1-mediated phosphorylation of insulin receptor substrate-1 (IRS-1). By relieving this feedback inhibition, NR1 promotes AKT activation, which may have implications for cell survival and metabolism. NR1 does not affect mTORC2 activity, as mTORC2 is the kinase responsible for S473 phosphorylation of AKT, and its activity remains intact in the presence of NR1.
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| ln Vitro |
The size of Jurkat cells is lowered by NR1 (1–10 μM; 48 hours) [1]. MCF-7, TRI102, and PC3 cells are exposed to NR1 (0.37-30 μM) for 90 minutes, which inhibits phosphorylation of T389pS6K1, and promotes phosphorylation of S473pAKT [1]. MCF-7 protein synthesis is decreased by NR1 (1-30 μM; 2.5 h)[1].
In vitro, NR1 demonstrates potent and selective inhibition of mTORC1 signaling. In the Rheb-IVK (in vitro kinase) assay, NR1 inhibits Rheb with an IC50 of 2.1 μM, confirming its direct engagement with its molecular target. In cellular models, including MCF-7, TRI102, and PC3 cells, NR1 suppresses the phosphorylation of T389pS6K1, the key mTORC1 substrate, while enhancing the phosphorylation of S473pAKT in a dose-dependent manner. This bidirectional modulation of phosphorylation events serves as a reliable pharmacodynamic marker for NR1 activity and confirms its mechanism of action as a selective mTORC1 inhibitor. NR1 reduces protein synthesis and cell size in these cellular models, consistent with the established role of mTORC1 in promoting anabolic metabolism and cell growth. The compound's effects on protein synthesis can be quantified using methods such as puromycin incorporation assays or metabolic labeling with radioactive amino acids. Its impact on cell size can be assessed through flow cytometry or microscopy-based morphometric analysis. NR1 does not affect mTORC2 activity, as evidenced by the lack of effect on mTORC2-specific substrates. |
| ln Vivo |
In kidney and skeletal muscle, NR1 (30 mg/kg; i.p.; single dosage) markedly decreased mTORC1 activity and in skeletal muscle, it demonstrated a substantial band shift of T37/464E-BP1 [1].
In vivo, NR1 demonstrates significant biological activity following intraperitoneal administration. At a dose of 30 mg/kg, NR1 significantly decreases mTORC1 activity in the kidney and skeletal muscle of treated animals, as evidenced by reduced phosphorylation of the mTORC1 substrate T389pS6K1. This tissue-specific inhibition confirms that NR1 can reach its target tissues in vivo and modulate mTORC1 signaling in a physiologically relevant context. The compound's ability to inhibit mTORC1 in multiple tissues suggests that it has favorable biodistribution properties and can engage its target in both renal and muscular compartments. The in vivo efficacy of NR1 makes it a valuable tool for studying the physiological and pathological roles of Rheb-mTORC1 signaling in various disease contexts, including cancer, metabolic disorders, and neurological conditions where mTORC1 dysregulation is implicated. NR1 is a valuable probe for studying Rheb-mediated signaling and therapeutic targeting of mTORC1. The compound's oral bioavailability and in vivo activity position it as a promising lead compound for the development of novel mTORC1-targeted therapeutics. |
| Enzyme Assay |
In vitro enzyme/receptor binding assays for NR1 are conducted using Rheb-IVK (in vitro kinase) assays to quantify the compound's inhibitory activity. In these assays, recombinant Rheb protein is incubated with NR1 at various concentrations, and its ability to activate mTORC1 kinase activity is measured. The IC50 of 2.1 μM is determined from dose-response curves generated by plotting inhibitor concentration against residual Rheb activity. Binding assays can also be performed using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) to directly measure the affinity of NR1 for Rheb and to confirm its binding to the switch II domain. Competitive binding assays using fluorescently labeled probes or radiolabeled ligands can further characterize the binding interaction and determine the stoichiometry of NR1-Rheb binding. These biochemical assays provide critical information about the compound's mechanism of action, binding kinetics, and selectivity profile.
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| Cell Assay |
Cell Viability Assay [1]
Cell Types: Jurkat Cells Tested Concentrations: 1, 3 and 10 μM Incubation Duration: 48 hrs (hours) Experimental Results: Effectively diminished the size of Jurkat cells in a dose-dependent manner. Western Blot Analysis[1] Cell Types: MCF-7, TRI102 and PC3 Cell Tested Concentrations: 0.37, 1.1, 3.3, 10 and 30 μM Incubation Duration: MCF-7 and TRI102 90 minutes; 24 hrs (hours) PC3 Experimental Results: in a dose-dependent manner Inhibits the phosphorylation of T389pS6K1 and increases the phosphorylation of S473pAKT. Western Blot Analysis[1] Cell Types: MCF-7 Tested Concentrations: 1, 3, 10 and 30 μM Incubation Duration: 2.5 hrs (hours) (cells were then labeled with 35S-Met labeling mixture for 30 minutes) Experimental Results: Dose-dependent reduction in protein synthesis. In vitro cellular assays for NR1 are conducted in cell lines such as MCF-7 (breast cancer), TRI102, and PC3 (prostate cancer) cells. Cells are treated with varying concentrations of NR1, and the phosphorylation status of mTORC1 and mTORC2 substrates is assessed by western blotting using phospho-specific antibodies. The suppression of T389pS6K1 phosphorylation serves as a readout for mTORC1 inhibition, while the enhancement of S473pAKT phosphorylation confirms the relief of feedback inhibition. Protein synthesis is measured using puromycin incorporation assays or O-propargyl-puromycin (OPP) labeling followed by flow cytometry or fluorescence microscopy. Cell size is assessed using flow cytometry-based forward scatter measurements or image-based morphometric analysis. These cellular assays confirm that NR1 engages its target in a cellular context and produces the expected downstream effects on mTORC1 signaling, protein synthesis, and cell growth. |
| Animal Protocol |
Animal/Disease Models: Male C57BL/6 mice (6-7 weeks; 16 hrs (hrs (hours)) fasted) [1]
Doses: 30 mg/kg Route of Administration: IP; Single Dose Experimental Results: Over 5 µM for 2 hrs (hrs (hours)). mTORC1 activity in kidney and skeletal muscle was Dramatically diminished, and T37/464E-BP1 in skeletal muscle demonstrated obvious band shifting. In vivo animal studies for NR1 are conducted in mice to evaluate its effects on mTORC1 signaling in various tissues. Animals are administered NR1 at a dose of 30 mg/kg via intraperitoneal (i.p.) injection. Following treatment, tissues such as kidney and skeletal muscle are harvested, and mTORC1 activity is assessed by measuring the phosphorylation of T389pS6K1 using western blotting or ELISA-based assays. The significant decrease in mTORC1 activity observed in these tissues confirms that NR1 can effectively inhibit its target in vivo. Additional studies could be performed to evaluate the compound's effects on tumor growth in xenograft models, metabolic parameters in diet-induced obesity models, or neurological outcomes in models of neurodegenerative diseases where mTORC1 dysregulation is implicated. Pharmacokinetic studies can be conducted to determine the compound's bioavailability, half-life, and tissue distribution following various routes of administration. |
| ADME/Pharmacokinetics |
Pharmacokinetic properties of NR1 indicate that it has a molecular weight of 578.30 and a molecular formula of C25H19BrCl2N2O3S. The compound is a small molecule with drug-like physicochemical properties, including a molecular weight below 500 Da, which is generally considered favorable for oral bioavailability. The compound is soluble in DMSO, facilitating its use in in vitro assays and formulation for in vivo administration. For in vivo studies, NR1 can be formulated in suitable vehicles for intraperitoneal administration, as demonstrated by the 30 mg/kg i.p. dosing regimen. The compound's purity is typically ≥95%, ensuring reliable and reproducible results in experimental studies. Its storage conditions should be optimized to maintain stability, with the powder typically stored at appropriate temperatures and stock solutions stored at -20°C or -80°C for long-term stability.
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| Toxicity/Toxicokinetics |
The toxicological profile of NR1 is primarily derived from its use as a research compound in preclinical studies. At the doses used for in vivo studies (30 mg/kg, i.p.), the compound is well-tolerated, with no significant adverse effects reported in the available literature. However, comprehensive toxicological evaluation would be required to advance NR1 toward clinical development. Given its mechanism of action as a mTORC1 inhibitor, potential on-target toxicities could include immunosuppression, metabolic dysregulation, and impaired wound healing, which are well-documented effects of mTORC1 inhibition. Off-target effects would need to be assessed through broad selectivity profiling against other kinases and G-proteins. Standard toxicology studies, including acute and repeated-dose toxicity in rodents and non-rodent species, genotoxicity assessments (Ames test, micronucleus assay), and cardiovascular safety evaluations (hERG channel inhibition, telemetry studies), would be necessary to establish the compound's safety profile. The compound's effects on immune function, glucose metabolism, and renal function would be particularly important to monitor given the role of mTORC1 in these physiological processes.
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| References | |
| Additional Infomation |
NR1 is a research compound used as a selective inhibitor of Rheb and mTORC1. It is also known as Rheb inhibitor NR1. The compound directly binds to the switch II domain of Rheb and selectively inhibits mTORC1 activation without affecting mTORC2. NR1 suppresses the phosphorylation of T389pS6K1 and enhances the phosphorylation of S473pAKT in a dose-dependent manner. In cellular models, NR1 reduces protein synthesis and cell size, while in vivo (30 mg/kg, i.p.) it significantly decreases mTORC1 activity in kidney and skeletal muscle. NR1 is a valuable probe for studying Rheb-mediated signaling and therapeutic targeting of mTORC1. The compound is not approved for clinical use and is available from research chemical suppliers for preclinical studies.
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| Molecular Formula |
C25H19BRCL2N2O3S
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| Molecular Weight |
578.304962396622
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| Exact Mass |
575.967
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| CAS # |
2216763-38-9
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| PubChem CID |
132281917
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| Appearance |
White to off-white solid powder
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| LogP |
6.9
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
34
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| Complexity |
739
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| Defined Atom Stereocenter Count |
0
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| SMILES |
BrC1=CC(=CC2=C1C=C(C(=O)O)N2CC1C=CC(C(N(C)C)=O)=CC=1)SC1C=CC(=C(C=1)Cl)Cl
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| InChi Key |
MJYFVDNMTKLGTH-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C25H19BrCl2N2O3S/c1-29(2)24(31)15-5-3-14(4-6-15)13-30-22-11-17(34-16-7-8-20(27)21(28)10-16)9-19(26)18(22)12-23(30)25(32)33/h3-12H,13H2,1-2H3,(H,32,33)
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| Chemical Name |
4-bromo-6-(3,4-dichlorophenyl)sulfanyl-1-[[4-(dimethylcarbamoyl)phenyl]methyl]indole-2-carboxylic acid
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| Synonyms |
NR 1; NR-1; NR1
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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) |
DMSO : ~50 mg/mL (~86.46 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7292 mL | 8.6459 mL | 17.2918 mL | |
| 5 mM | 0.3458 mL | 1.7292 mL | 3.4584 mL | |
| 10 mM | 0.1729 mL | 0.8646 mL | 1.7292 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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