FK506-binding protein 51 (FKBP51). [2]

In 51KO cells, SAFit2 administration had no anti-FKBP51 panoramic effect, but it did raise the expression of pAKT2 (soleus and EDL ablated) and pAS160 (EDL ablated) in WT cells. Moreover, following SAFit2 administration, GLUT4 rose in the membrane fraction of primary EDL-expressing myotubes in WT cells but not in 51KO animals [2].

---

In primary EDL myotubes collected from WT mice, SAFit2 treatment (0.6 μM overnight) increased the expression of pAKT2 and pAS160 in both soleus and EDL muscle cells, and increased GLUT4 expression at the plasma membrane. [2]
In primary EDL myotubes from WT mice, SAFit2 treatment (0.6 μM overnight) increased 2-deoxyglucose uptake under both non-insulin and insulin-stimulated conditions. [2]
In primary EDL myotubes collected from 51KO (FKBP51 knockout) mice, SAFit2 had no effect on pAKT2, pAS160, GLUT4 membrane expression, or glucose uptake, demonstrating the specificity of SAFit2 for FKBP51. [2]
In C2C12 myotubes, SAFit2 (concentration not specified) disrupted the interaction between FKBP51 and AS160, as demonstrated by co-immunoprecipitation. [2]

Under both control and high-fat diet (HFD) circumstances, it was discovered that SAFit2 led to weight loss for 30 days. When SAFit2 was applied an hour before testing, no changes in anxiety-related behaviors were seen. SAFit2 significantly increases phosphorylated AKT2 and AS160 in the EDL as well as increases GLUT4 expression on the soleus muscle membrane [2]. The elevated plus maze (EPM) significantly increased the open-arm time (z=-2.183, p<0.05) in response to the induction of treatment injection 16 hours prior to testing, indicating that SAFit2 induced an anxiolytic phenotype. The study found that the SAFit2 treatment led to a significant increase in both the delay time (z=-2-265, p<0.05) and travel distance (t(20)=-2.371, p<0.05) in the illuminated compartment.

---

Acute SAFit2 treatment: Male C57BL/6 mice received a single subcutaneous injection of slow-release formulated gel containing SAFit2 (2 mg) or vehicle. After 48 hours, SAFit2-treated mice showed improved glucose tolerance in a glucose tolerance test following an overnight fast. [2]
Sub-chronic SAFit2 treatment: Male C57BL/6 mice received twice-daily intraperitoneal injections of SAFit2 (20 mg/kg) or vehicle for 10 days. On day 8 of treatment, SAFit2-treated mice showed improved glucose tolerance. Body weight was not significantly affected by SAFit2 treatment at this time point. [2]
Chronic SAFit2 treatment: Male C57BL/6 mice on high-fat diet received twice-daily intraperitoneal injections of SAFit2 (20 mg/kg) or vehicle for 30 days. SAFit2 treatment protected against HFD-induced weight gain and improved glucose tolerance (assessed on day 25). [2]
Six hours after a single intraperitoneal injection of SAFit2 (20 mg/kg), mice showed increased phosphorylation of AKT2 and AS160 in EDL muscle, and increased GLUT4 expression at the plasma membrane in soleus muscle, under both insulin-stimulated (0.70 IU/kg insulin, 5 min before tissue collection) and non-insulin (saline) conditions. [2]
In 30-day vehicle-treated and SAFit2-treated mice (20 mg/kg twice daily), co-immunoprecipitation from soleus and EDL muscle lysates revealed that SAFit2 treatment increased binding between AKT2 and AS160, while simultaneously decreasing binding between FKBP51 and AS160. SAFit2 had no effect on the binding between AKT2 and PHLPP1. [2]

Primary EDL myotube culture: Satellite cells were collected from soleus and EDL muscles of 4- to 8-week-old WT and 51KO mice, differentiated for 5 days, and used for experiments. [2]
C2C12 myotube culture: C2C12 myoblasts were maintained in DMEM with 10% FBS, differentiated with 2% horse serum for 3 days, and used for experiments. [2]
Glucose uptake assay: Cells were serum-starved for 4 hours, incubated in KRH buffer, stimulated with insulin (100 nM) or left unstimulated for 1 hour, then incubated with 100 μM 2-deoxy-D-[1,2-³H]glucose (2 μCi/mL) for 4 minutes. Reactions were terminated with cytochalasin B, and incorporated radioactivity was measured by liquid scintillation counting, normalized to total protein by BCA assay. For SAFit2 experiments, a toxicity assay was initially performed to determine the appropriate concentration; based on LD15, cells were incubated with 0.6 μM SAFit2 or DMSO overnight before glucose uptake induction. [2]
GLUT4 membrane localization: Primary EDL myotubes were treated with 0.6 μM SAFit2 or DMSO overnight, serum-starved for 4 hours, and then plasma membrane fractions were prepared. GLUT4 expression was detected by Western blot and normalized to Na,K-ATPase and total GLUT4. [2]
Co-immunoprecipitation: Protein extracts (500 μg) from soleus muscle, EDL, and eWAT of vehicle-treated and SAFit2-treated HFD-fed mice were incubated overnight with 2 μg of AKT2 or FKBP51 antibody. Protein-antibody complexes were eluted and analyzed by Western blot against AKT2, FKBP51, PHLPP1, and AS160. [2]

Animals: Male C57BL/6 mice (3-4 months old) were used for pharmacological studies; male FKBP51 knockout (51KO) mice and wild-type littermates (3-4 months old) were used for genetic studies. Mice were singly housed on a 12:12 h light/dark cycle with controlled temperature (22 ± 2°C) and humidity (55 ± 5%). [2]
Acute SAFit2 treatment: Mice received a single subcutaneous injection (between the shoulders) of a slow-release vesicular phospholipid gel (VPG) formulation containing 2 mg SAFit2 or vehicle. VPGs were composed of 50% (m/m) egg-lecithin and 10 mM PBS (pH 7.4). SAFit2 was encapsulated by direct incorporation. After 48 hours, a glucose tolerance test was performed following an overnight fast. [2]
Sub-chronic SAFit2 treatment: Mice received twice-daily intraperitoneal injections of SAFit2 (20 mg/kg) or vehicle for 10 days. SAFit2 was solubilized in vehicle containing 4% ethanol, 5% Tween80, and 5% PEG400 in 0.9% saline. On day 8, a glucose tolerance test was performed. [2]
Chronic SAFit2 treatment: Four weeks before treatment onset, mice were placed on control diet or high-fat diet (HFD, 58% kcal from fat). Mice then received twice-daily intraperitoneal injections of SAFit2 (20 mg/kg) or vehicle for 30 days. Body weight and food intake were measured daily. Glucose tolerance test was performed on day 25; insulin tolerance test on day 29. Animals were killed on day 31; tissues were collected for analysis. [2]
Glucose tolerance test (GTT): Mice were fasted overnight, then received an intraperitoneal glucose injection (2 g/kg body weight). Blood glucose was measured at 0, 15, 30, 60, 90, and 120 minutes. [2]
Insulin tolerance test (ITT): Mice were fasted for 4 hours, then received an intraperitoneal insulin injection (0.70 IU/kg body weight). Blood glucose was measured at 0, 15, 30, 60, and 120 minutes. [2]

---

Animals: Male C57BL/6 mice (3-4 months old) were used for pharmacological studies; male FKBP51 knockout (51KO) mice and wild-type littermates (3-4 months old) were used for genetic studies. Mice were singly housed on a 12:12 h light/dark cycle with controlled temperature (22 ± 2°C) and humidity (55 ± 5%). [2]
Acute SAFit2 treatment: Mice received a single subcutaneous injection (between the shoulders) of a slow-release vesicular phospholipid gel (VPG) formulation containing 2 mg SAFit2 or vehicle. VPGs were composed of 50% (m/m) egg-lecithin and 10 mM PBS (pH 7.4). SAFit2 was encapsulated by direct incorporation. After 48 hours, a glucose tolerance test was performed following an overnight fast. [2]
Sub-chronic SAFit2 treatment: Mice received twice-daily intraperitoneal injections of SAFit2 (20 mg/kg) or vehicle for 10 days. SAFit2 was solubilized in vehicle containing 4% ethanol, 5% Tween80, and 5% PEG400 in 0.9% saline. On day 8, a glucose tolerance test was performed. [2]
Chronic SAFit2 treatment: Four weeks before treatment onset, mice were placed on control diet or high-fat diet (HFD, 58% kcal from fat). Mice then received twice-daily intraperitoneal injections of SAFit2 (20 mg/kg) or vehicle for 30 days. Body weight and food intake were measured daily. Glucose tolerance test was performed on day 25; insulin tolerance test on day 29. Animals were killed on day 31; tissues were collected for analysis. [2]
Glucose tolerance test (GTT): Mice were fasted overnight, then received an intraperitoneal glucose injection (2 g/kg body weight). Blood glucose was measured at 0, 15, 30, 60, 90, and 120 minutes. [2]
Insulin tolerance test (ITT): Mice were fasted for 4 hours, then received an intraperitoneal insulin injection (0.70 IU/kg body weight). Blood glucose was measured at 0, 15, 30, 60, and 120 minutes. [2]

[1]. Rapid, Structure-Based Exploration of Pipecolic Acid Amides as Novel Selective Antagonists of the FK506-Binding Protein 51. J Med Chem. 2016 Mar 24;59(6):2410-22.

[2]. Stress-responsive FKBP51 regulates AKT2-AS160 signaling and metabolic function. Nat Commun. 2017 Nov 23;8(1):1725.

[3]. Pharmacological Inhibition of the Psychiatric Risk Factor FKBP51 Has Anxiolytic Properties. J Neurosci. 2015 Jun 17;35(24):9007-16.

SAFit2 is a selective antagonist of FK506-binding protein 51 (FKBP51). It was developed as a tool to investigate the role of FKBP51 in various physiological processes. This study demonstrates that SAFit2 improves glucose tolerance and insulin sensitivity in mice by enhancing AKT2-AS160 signaling and glucose uptake in skeletal muscle. Mechanistically, SAFit2 disrupts the interaction between FKBP51 and AS160, while enhancing the binding between AKT2 and AS160, leading to increased GLUT4 translocation to the plasma membrane and increased glucose uptake. The effects of SAFit2 are specific to FKBP51, as they are absent in FKBP51 knockout mice and primary cells. These findings suggest that FKBP51 antagonism may represent a therapeutic strategy for treating type 2 diabetes and obesity. [2]

C46H62N2O10

802.991894245148

802.44

1643125-33-0

86277887

Off-white to pink solid powder

8.2

0

11

19

58

1200

3

O=C([C@H](C1C=C(C(=C(C=1)OC)OC)OC)C1CCCCC1)N1CCCC[C@H]1C(=O)O[C@@H](C1C=CC=C(C=1)OCCN1CCOCC1)CCC1C=CC(=C(C=1)OC)OC

ZDBWLRLGUBSLPG-FDHYQTMZSA-N

InChI=1S/C46H62N2O10/c1-51-39-20-18-32(28-40(39)52-2)17-19-38(34-14-11-15-36(29-34)57-27-24-47-22-25-56-26-23-47)58-46(50)37-16-9-10-21-48(37)45(49)43(33-12-7-6-8-13-33)35-30-41(53-3)44(55-5)42(31-35)54-4/h11,14-15,18,20,28-31,33,37-38,43H,6-10,12-13,16-17,19,21-27H2,1-5H3/t37-,38+,43-/m0/s1

[(1R)-3-(3,4-dimethoxyphenyl)-1-[3-(2-morpholin-4-ylethoxy)phenyl]propyl] (2S)-1-[(2S)-2-cyclohexyl-2-(3,4,5-trimethoxyphenyl)acetyl]piperidine-2-carboxylate

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 : ~100 mg/mL (~124.53 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.11 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (2.59 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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (2.59 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.)