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| Targets |
The primary target of SAFit1 is FK506-binding protein 51 (FKBP51), a 51 kDa immunophilin that functions as a co-chaperone in the Hsp90 complex. FKBP51 is a key regulator of the glucocorticoid receptor (GR), a nuclear receptor that mediates the effects of cortisol. In the absence of cortisol, the GR is held in an inactive complex by Hsp90 and co-chaperones, including FKBP51. When cortisol binds, FKBP51 dissociates, and the receptor translocates to the nucleus to regulate gene transcription. FKBP51 acts as a negative regulator of GR signaling by reducing the receptor's affinity for cortisol and by promoting its dephosphorylation. By binding to FKBP51, SAFit1 disrupts its interaction with the Hsp90 complex, thereby increasing the sensitivity of the GR to cortisol and enhancing GR signaling. This is the opposite effect of FKBP51, which decreases GR sensitivity. The high selectivity of SAFit1 for FKBP51 over FKBP52 is critical because FKBP52 has the opposite effect on GR signaling, enhancing it. By selectively inhibiting FKBP51, SAFit1 allows researchers to specifically study the role of this isoform in GR signaling and its involvement in various physiological and pathological processes, including stress responses, depression, anxiety, and metabolic disorders.
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| ln Vitro |
Primary hippocampal neurons and two neuronal cell lines (N2a and SH-SY5Y) are both responsive to the effective neurite outgrowth stimulant SAFit1 (1-1000 nM). A broad range of concentrations is where SAFit1 is active [1].
In vitro studies have characterized SAFit1 as a highly potent and selective inhibitor of FKBP51. Its activity is typically measured using competitive binding assays. In these assays, a labeled FK506 or a fluorescent probe that binds to the FKBP51 active site is used. SAFit1 is added at increasing concentrations, and its ability to displace the labeled ligand is measured. The Ki, which is the inhibition constant, is determined from the competition curve. SAFit1 has a Ki of 4±0.3 nM for FKBP51. Its selectivity is demonstrated by testing it against FKBP52, where it shows a Ki of >50,000 nM, representing a >12,500-fold selectivity. In cell-based assays, SAFit1 has been shown to enhance glucocorticoid receptor signaling. This is typically measured by treating cells with a glucocorticoid (e.g., dexamethasone) in the presence or absence of SAFit1 and then measuring the expression of GR target genes using quantitative real-time PCR or reporter gene assays. SAFit1 has been shown to potentiate the transcriptional activity of the GR. These in vitro studies confirm that SAFit1 is a potent and selective tool for studying FKBP51 function. |
| ln Vivo |
In vivo studies have demonstrated that SAFit1 is biologically active and can modulate glucocorticoid receptor signaling in animal models. For example, in mouse models of stress or depression, SAFit1 has been shown to produce antidepressant-like effects. The mechanism is believed to involve the enhancement of GR signaling in the brain, which can help to normalize the stress response. In metabolic studies, SAFit1 has been shown to improve glucose tolerance and insulin sensitivity, suggesting a role for FKBP51 in metabolic regulation. These in vivo studies are crucial for validating FKBP51 as a therapeutic target for neuropsychiatric and metabolic disorders. The compound's high selectivity for FKBP51 is expected to minimize off-target effects, making it a valuable tool for preclinical research. SAFit1 is typically administered to animals by injection, and its effects are assessed by measuring behavioral, biochemical, and physiological endpoints. These studies have provided important insights into the role of FKBP51 in health and disease.
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| Enzyme Assay |
The in vitro binding assays for SAFit1 are designed to measure its affinity for FKBP51 and its selectivity over FKBP52. A common assay is a fluorescence polarization (FP) competition assay. In this assay, a fluorescently labeled probe that binds to the FKBP51 active site (e.g., a fluorescein-labeled FK506 analog) is mixed with recombinant FKBP51 protein. The binding of the probe to the protein results in an increase in fluorescence polarization. SAFit1 is then added at increasing concentrations, and the decrease in fluorescence polarization is measured. The decrease in polarization indicates that SAFit1 is competing with the probe for binding to FKBP51. The data is used to calculate the Ki. Alternatively, a radioligand binding assay can be used, where a radiolabeled FK506 analog is used instead of a fluorescent probe. For selectivity testing, the same assay is performed using FKBP52 protein. The Ki for FKBP51 is 4±0.3 nM, and the Ki for FKBP52 is >50,000 nM, demonstrating the exceptional selectivity of SAFit1. These assays are standard for characterizing FKBP51 inhibitors.
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| Cell Assay |
In vitro cell-based assays for SAFit1 are used to study its effects on glucocorticoid receptor (GR) signaling. A common assay is the reporter gene assay. In this assay, cells (e.g., HEK293 cells) are transfected with a plasmid containing a luciferase reporter gene under the control of a glucocorticoid response element (GRE). The cells are then treated with a glucocorticoid (e.g., dexamethasone) in the presence or absence of SAFit1. After a period of incubation, the cells are lysed, and the luciferase activity is measured. An increase in luciferase activity in the presence of SAFit1 indicates that it is enhancing GR signaling. Another cell-based assay is the measurement of endogenous GR target gene expression. Cells are treated with dexamethasone with or without SAFit1, and the mRNA levels of GR target genes (e.g., FKBP5, GILZ) are measured by quantitative real-time PCR. An increase in the expression of these genes in the presence of SAFit1 confirms that it potentiates GR signaling. These cell-based assays are crucial for confirming that SAFit1's binding to FKBP51 translates into a functional effect on GR signaling.
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| Animal Protocol |
In vivo animal experiments for SAFit1 are typically conducted in mouse models. For studying its effects on stress and depression, a common model is the forced swim test or the tail suspension test. In these tests, mice are placed in an inescapable situation (a cylinder of water or suspended by their tail), and their immobility time is measured. A decrease in immobility time is interpreted as an antidepressant-like effect. In a typical protocol, SAFit1 is administered to mice (e.g., intraperitoneally) 30-60 minutes before the test, and the immobility time is compared to a vehicle control group. For studying its effects on metabolism, a common model is the high-fat diet-induced obesity model. Mice are fed a high-fat diet to induce obesity and insulin resistance, and then they are treated with SAFit1. Parameters such as body weight, glucose tolerance (by glucose tolerance test), and insulin sensitivity are measured. These in vivo studies are essential for demonstrating the therapeutic potential of SAFit1 and for understanding the role of FKBP51 in these physiological processes.
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| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of SAFit1 have been characterized in preclinical studies. SAFit1 has a molecular weight of 747.87 g/mol and a molecular formula of C42H53NO11. It is a large, lipophilic molecule, which may affect its oral bioavailability. For research use, SAFit1 is typically supplied as a solid and is soluble in DMSO. Its stability is ensured by storing it under recommended conditions (typically -20°C). In animal studies, it is often administered by intraperitoneal (i.p.) or intravenous (i.v.) injection to ensure consistent delivery. Its half-life and clearance would determine the dosing frequency. A comprehensive PK profile is essential for interpreting in vivo efficacy studies. As a research compound, its PK properties are well-characterized for use in animal models, but it is not developed for human use.
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| Toxicity/Toxicokinetics |
Detailed toxicity data for SAFit1 is not typically provided in product descriptions, as it is a research compound. However, its high selectivity for FKBP51 over FKBP52 (>12,500-fold) suggests that it may have a favorable safety profile. The primary on-target effects of SAFit1 would be related to the enhancement of glucocorticoid receptor signaling, which could have physiological consequences, such as increased cortisol sensitivity. However, these are the effects that are being studied. Off-target toxicity is expected to be low due to its high selectivity. In preclinical studies, the safety of SAFit1 would be assessed by monitoring body weight, clinical signs, and organ histology in treated animals. Any use in vivo requires careful consideration of the dose and route of administration. As a research tool, it is handled with standard laboratory safety precautions.
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| References | |
| Additional Infomation |
SAFit1 is a research compound and is not approved for any clinical use. It is a highly potent and selective inhibitor of FKBP51, a co-chaperone of the Hsp90 complex that regulates glucocorticoid receptor (GR) signaling. Its mechanism of action involves binding to FKBP51 and inhibiting its function, which leads to enhanced GR signaling. This makes SAFit1 a valuable tool for studying the role of FKBP51 in a variety of physiological and pathological processes, including stress biology, neuropsychiatric disorders (depression, anxiety), and metabolic diseases (obesity, diabetes). Its exceptional selectivity for FKBP51 over FKBP52 (>12,500-fold) is a key feature that allows for the specific study of this isoform without confounding effects. SAFit1 is widely used in academic and pharmaceutical research to validate FKBP51 as a therapeutic target. It is available for research purposes only and is not intended for human use.
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| Molecular Formula |
C42H53NO11
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| Molecular Weight |
747.870333433151
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| Exact Mass |
747.361
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| CAS # |
1643125-32-9
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| PubChem CID |
86277875
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| Appearance |
White to yellow solid powder
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| LogP |
8.2
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
18
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| Heavy Atom Count |
54
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| Complexity |
1150
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| Defined Atom Stereocenter Count |
3
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| SMILES |
COC1=C(C=C(C=C1)CC[C@H](C2=CC(=CC=C2)OCC(=O)O)OC(=O)[C@@H]3CCCCN3C(=O)[C@@H](C4CCCCC4)C5=CC(=C(C(=C5)OC)OC)OC)OC
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| InChi Key |
OEQZPFWOEOOISR-AKTKKGGCSA-N
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| InChi Code |
InChI=1S/C42H53NO11/c1-48-34-20-18-27(22-35(34)49-2)17-19-33(29-14-11-15-31(23-29)53-26-38(44)45)54-42(47)32-16-9-10-21-43(32)41(46)39(28-12-7-6-8-13-28)30-24-36(50-3)40(52-5)37(25-30)51-4/h11,14-15,18,20,22-25,28,32-33,39H,6-10,12-13,16-17,19,21,26H2,1-5H3,(H,44,45)/t32-,33+,39-/m0/s1
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| Chemical Name |
2-[3-[(1R)-1-[(2S)-1-[(2S)-2-cyclohexyl-2-(3,4,5-trimethoxyphenyl)acetyl]piperidine-2-carbonyl]oxy-3-(3,4-dimethoxyphenyl)propyl]phenoxy]acetic acid
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| Synonyms |
SAFit1 SAF-it-1 SA-Fit-1
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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 : ≥ 300 mg/mL (~401.14 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (2.78 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 20.8 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (2.78 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3371 mL | 6.6857 mL | 13.3713 mL | |
| 5 mM | 0.2674 mL | 1.3371 mL | 2.6743 mL | |
| 10 mM | 0.1337 mL | 0.6686 mL | 1.3371 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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