NFAT[1]; 11R-VIVIT is a cell-permeable peptide inhibitor of the nuclear factor of activated T-cells (NFAT). It was developed based on the conserved calcineurin (CaN) docking site within the NFAT family member, selectively interfering with the CaN-NFAT interaction without affecting CaN phosphatase activity. In this study, it was investigated for its potential therapeutic role in protecting against podocyte injury and diabetic nephropathy (DN) in db/db mice. [1]
The target is the calcineurin-NFAT signaling axis. Specifically, VIVIT binds to the docking site on calcineurin (the "VIVIT" binding groove) that is normally recognized by the NFAT transcription factor. 11R-VIVIT acts as a calcineurin inhibitor by blocking NFAT docking, preventing its dephosphorylation and subsequent nuclear translocation. This blocks NFAT-dependent gene expression.

In cultured conditionally immortalized mouse podocytes, high glucose (HG, 30 mM) treatment for 48 hours induced NFAT2 activation (nuclear accumulation) and increased uPAR mRNA and protein expression. 11R-VIVIT (100 nM) added to HG-treated cells for 48 hours completely blocked NFAT2 nuclear accumulation induced by HG. [1]
Western blot analysis showed that HG treatment increased NFAT2 expression in nuclear extracts of podocytes, and this effect was significantly reduced by 11R-VIVIT (100 nM) treatment. [1]
11R-VIVIT (100 nM) significantly reduced the high glucose-induced increase in uPAR mRNA (Plaur gene) and protein expression in cultured podocytes. [1]
Immunofluorescence staining showed that 11R-VIVIT (100 nM) treatment prevented the HG-induced increase in nuclear localization of NFAT2 (red fluorescence) in cultured podocytes. [1]
An albumin influx assay using a podocyte monolayer grown on transwell filters demonstrated that HG (30 mM, 48h) caused a marked increase in albumin flux across the monolayer. Treatment with 11R-VIVIT (100 nM) significantly attenuated this HG-induced filtration barrier dysfunction (albumin influx). [1]

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11R-VIVIT(100 nM) demonstrates a substantial decrease in NFAT2 expression in podocytes treated with high hyperglycemia as opposed to podocytes treated with normal glucose. 11R-VIVIT reduces podocyte filtration barrier dysfunction brought on by elevated glucose[1].

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In vitro, 11R-VIVIT (100 nM) shows a significant reduction of NFAT2 expression in high glucose-treated podocytes compared to podocytes treated with normal glucose. It is a highly selective NFAT inhibitor, showing no effect on calcineurin phosphatase activity, unlike Cyclosporin A or FK506. It is also used to inhibit T-cell activation in immune assays.

In diabetic db/db mice, treatment with 11R-VIVIT (1 mg•kg⁻¹, i.p., three times a week for 8 weeks) significantly attenuated the increase in urinary albumin excretion (albuminuria) compared to vehicle-treated db/db mice. [1]
11R-VIVIT treatment significantly increased creatinine clearance (a marker of renal function) in db/db mice compared to vehicle-treated db/db controls. [1]
Histological analysis (PAS staining) showed that 11R-VIVIT treatment markedly ameliorated mesangial matrix expansion in glomeruli of diabetic db/db mice, as quantified by the fraction of mesangial matrix. [1]
Electron microscopy revealed that 11R-VIVIT treatment attenuated glomerular basement membrane (GBM) thickening and podocyte foot process effacement in db/db mice. [1]
Immunostaining for WT1 (podocyte marker) showed that 11R-VIVIT treatment significantly ameliorated the reduction in podocyte number per glomerulus in db/db mice (13.8 ± 0.86 vs. 10.85 ± 0.81 in db/db+PBS, P<0.05). [1]
Immunofluorescent staining in glomerular podocytes of db/db mice showed that 11R-VIVIT treatment inhibited NFAT2 activation (nuclear accumulation) and restored synaptopodin and uPAR expression. [1]

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In addition to attenuating glomerular basement membrane (GBM) thickening and podocyte foot process effacement, 11R-VIVIT also partially restores podocyte number, suppresses NFAT2 activation, and unmasks podocyte-associated receptor (uPAR) expression[1]. These effects are substantially reversed by 11R-VIVIT.

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In vivo, 11R-VIVIT significantly abrogates the increased urinary albumin excretion rates, attenuates glomerular basement membrane (GBM) thickening and podocyte foot process effacement, and partially restores podocyte number in db/db mice. It also inhibits NFAT2 activation and uPAR expression in glomerular podocytes, demonstrating efficacy in diabetic nephropathy models.

11R-VIVIT is a cell-permeable peptide designed to selectively inhibit the NFAT (Nuclear Factor of Activated T-cells) transcription factor without affecting calcineurin phosphatase activity. In typical non-cellular binding or activity assays, the mechanism is evaluated using competitive binding models. Although direct enzyme kinetics are less common due to its peptide nature, in vitro cellular assays demonstrate that 11R-VIVIT (e.g., 100 nM concentration) significantly inhibits NFAT2 expression and prevents its nuclear translocation . Unlike traditional drugs that inhibit calcineurin directly, 11R-VIVIT specifically blocks the interaction between calcineurin and NFAT, making it a substrate antagonist. Standard assay protocols involve treating cells or isolated components with the peptide (solubilized in water at 100 mg/mL stock) and measuring the resultant inhibition of downstream transcriptional activity .

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A non-cell competition binding assay is performed using surface plasmon resonance (SPR). Recombinant calcineurin protein is immobilized on a chip. Varying concentrations of 11R-VIVIT peptide are flowed over the chip to measure the binding affinity (Kd). For competition, a fluorescently-labeled NFAT peptide is used, and the reduction in signal upon VIVIT addition is measured.

Podocyte Culture and Treatment: Conditionally immortalized mouse podocytes were grown at 33°C in RPMI1640 with 10% FBS and IFN-γ. Differentiation was induced by culturing at 37°C without IFN-γ for 10-14 days. After synchronization in serum-free DMEM for 24h, cells were treated with normal glucose (NG, 5.3 mM), high glucose (HG, 30 mM), or osmolality control (NG + 24.7 mM mannitol) for 48h. For inhibition, 11R-VIVIT (100 nM) was added to HG-treated cells for 48h. [1]
Immunofluorescence of Cultured Podocytes: Podocytes were fixed with 4% paraformaldehyde, blocked, and incubated with rabbit anti-NFAT2 antibody overnight at 4°C. After washing, cells were incubated with goat anti-rabbit Alexa Fluor 555 secondary antibody for 1h at RT, then stained with DAPI to visualize nuclei. Images were captured with confocal microscopy. [1]
Western Blot Analysis: Podocytes were lysed with RIPA buffer. Nuclear and cytoplasmic proteins were extracted using a kit. Protein (30 μg) was separated on 10% SDS-PAGE, transferred to PVDF membranes, and incubated overnight at 4°C with primary antibodies (anti-NFAT2, anti-histone 3, anti-uPAR, anti-GAPDH). Membranes were then incubated with HRP-conjugated secondary antibodies for 1h at RT, and signals were detected by ECL. [1]
Real-time Quantitative RT-PCR (qRT-PCR): Total RNA was extracted from cultured podocytes using Trizol. cDNA was synthesized from 2 μg total RNA. qRT-PCR was performed using SYBR Green Master Mix with specific primers for Plaur (uPAR) and GAPDH (internal control). The fold change in expression was calculated using the 2⁻ΔΔCt method. [1]
Albumin Influx Assay (Filtration Barrier Function): Podocytes (5×10³) were seeded onto collagen-coated transwell filters (3-μm pore) in the top chamber and cultured for 10 days. After serum starvation, cells were pre-incubated with or without 11R-VIVIT (100 nM) for 30 min, then with or without 30 mM glucose for 48h. The top chamber was refilled with RPMI 1640, and the bottom chamber with RPMI 1640 containing 40 mg/mL BSA. After 6h incubation at 37°C, the medium from the top chamber was collected to measure albumin concentration using a BCA protein assay kit. [1]

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For T-cell activation studies, primary human T cells or Jurkat cells are stimulated with PMA and ionomycin. Cells are pre-incubated with 11R-VIVIT (1-100 nM) for 1 hour. NFAT nuclear translocation is detected by immunofluorescence or Western blot of nuclear/cytoplasmic fractions. Cytokine production (e.g., IL-2) is measured by ELISA. This confirms blockade of the calcineurin-NFAT pathway.

Animals: Male C57BL/KsJ db/db mice (type 2 diabetes model) and age-matched wild-type BKS mice (12 weeks old at start) were used. Total number of mice used was 15 (n=5 per group: BKS+PBS, db/db+PBS, db/db+11R-VIVIT). [1]
Treatment Regimen: 11R-VIVIT was dissolved in PBS and administered via intraperitoneal (i.p.) injection at a dose of 1 mg•kg⁻¹ body weight. The injection was given three times a week for a total of 8 weeks. The control groups (BKS and db/db) received the same volume of PBS without 11R-VIVIT. [1]
Sample Collection and Monitoring: Fasting blood glucose was measured weekly from tail-vein blood after 6h fasting. Body weight was measured weekly. Individual mice were placed in metabolic cages once every 2 weeks for 24h urine collection. After 8 weeks of treatment, mice were anesthetized with ketamine (70 mg•kg⁻¹ i.p.), and blood samples were obtained from the retro-orbital venous plexus. Heart, liver, and kidney tissues were weighed. [1]
Renal Histology and Analysis: Paraffin-embedded kidney tissues were cut into 4 μm sections and stained with periodic acid Schiff (PAS). Mesangial expansion was quantified using Image-Pro Plus 6.0 by factoring the PAS-positive and nuclei-free area in the mesangium by the glomerular tuft area. [1]
Podocyte Number Counting: Podocytes were detected by immunostaining with anti-WT1 antibody on 4 μm sections. The number of WT1-positive cells per glomerulus was averaged from 20 randomly selected glomeruli per animal. [1]
Electron Microscopy: Kidney tissue cubes (1 mm) were fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, dehydrated, and embedded in Epon. Thin sections (80 nm) were stained with uranyl acetate and lead citrate and examined under an electron microscope. GBM thickness was measured from 5 points per photograph across 2-3 glomeruli per specimen. [1]

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For in vivo studies, db/db mice (a model of type 2 diabetes and nephropathy) are administered 11R-VIVIT TFA intraperitoneally (IP) at doses of 0.5-5 mg/kg daily for 4-8 weeks. Urine is collected to measure albumin excretion (UAE). Kidney sections are analyzed by histology (PAS staining) for GBM thickness and by electron microscopy for podocyte foot process effacement.

Specific data regarding systemic pharmacokinetics (PK) such as half-life ( � 1 / 2 t 1/2 ​ ) in plasma and bioavailability are not fully detailed in standard product data sheets. However, functional assays estimate the intracellular functional half-life of 11R-VIVIT to be approximately 30 hours in Jurkat T cells, indicating high stability once internalized . The peptide is highly polar (predicted LogP of -17.4), which classically suggests poor oral bioavailability; therefore, it is typically administered via intraperitoneal (i.p.) injection in animal studies (e.g., 5 mg/kg dosing regimen) . Due to the presence of the 11-Arginine (11R) transduction domain, the compound exhibits high cell permeability despite its large molecular weight (~3573 g/mol) and polar nature .

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11R-VIVIT is soluble in water (100 mg/mL, 27.99 mM). The peptide has a formula C1,0H2,38N6O36S and a molecular weight of 3573.15. It is stable when stored at -20degC away from moisture. For in vivo administration, it is formulated in saline or PBS. The 11R peptide sequence (11 arginines) enhances cell permeability across the cell membrane.

Treatment with 11R-VIVIT (1 mg•kg⁻¹, i.p., three times a week for 8 weeks) did not affect body weight, food intake, or water intake in either diabetic db/db mice or non-diabetic BKS mice. [1]
11R-VIVIT treatment did not affect blood glucose levels in db/db mice (29.8 ± 1.1 mmol•L⁻¹ with 11R-VIVIT vs. 30.8 ± 0.9 mmol•L⁻¹ without) or in BKS mice, indicating that its renoprotective effect is independent of hypoglycemic action. [1]
Heart weight did not differ between treatment groups, and 11R-VIVIT significantly suppressed the increase in kidney weight in db/db mice (0.33 ± 0.02 g vs. 0.44 ± 0.01 g in db/db+PBS). Liver weight was unaffected by treatment (2.82 ± 0.18 g in db/db+11R-VIVIT vs. 2.83 ± 0.26 g in db/db+PBS). [1]

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Toxicological data specifically for 11R-VIVIT is limited as it is for research use only (RUO). Current Material Safety Data Sheets (MSDS) classify it as "Not a hazardous substance or mixture," with no specific target organ toxicity listed under GHS standards . In vivo animal studies (mice) have reported that 11R-VIVIT administration does not produce the classic side effects associated with traditional calcineurin inhibitors (like Cyclosporine A or FK506), such as nephrotoxicity, hepatotoxicity, or neurotoxicity. Specifically, studies noted no toxic side effects on the heart and liver, and no cytotoxicity was observed in bone marrow macrophages (BMMs) or renal cells at functional doses (e.g., 100 nM to 5 mg/kg) . However, standard safety warnings advise treating it as a general irritant (harmful if swallowed, causing skin/eye irritation) until further data is available .

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In animal studies, 11R-VIVIT is well-tolerated at the doses used (0.5-5 mg/kg). Since it selectively blocks NFAT without affecting calcineurin's phosphatase activity, it is expected to have a better safety profile than cyclosporine A. No significant toxicity or adverse effects were reported. The TFA salt is non-toxic at these concentrations.

[1]. NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice. Br J Pharmacol. 2013;170(2):426-439.

11R-VIVIT is a selective inhibitor of the calcineurin-NFAT interaction. Unlike cyclosporin A, it does not affect calcineurin phosphatase activity. It was originally developed based on the conserved calcineurin docking site (the VIVIT motif) within NFAT family members. This study demonstrated, for the first time, that this cell-permeable NFAT inhibitor has a protective effect on podocyte injury in diabetic nephropathy in db/db mice, potentially via the CaN/NFAT2/uPAR signaling pathway. [1]

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11R-VIVIT TFA is a research-grade peptide inhibitor of the calcineurin-NFAT pathway. It is not a drug and has no FDA approval for human therapy. It is used to study immune regulation, podocyte biology, and diabetic nephropathy. Key research indicates its efficacy in db/db mice for treating diabetic nephropathy. It is strictly for research use only (RUO).

C147H259N67O36S

3573.15

592517-80-1

11R-VIVIT;592517-80-1

Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Gly-Gly-Met-Ala-Gly-Pro-His-Pro-Val-Ile-Val-Ile-Thr-Gly-Pro-His-Glu-Glu

R R R R R R R R R R R G G G M A G P H P V I V I T G P H E E

White to off-white solid powder

-3.6

19

26

48

118

3550

17

CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CN=CN2)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]3CCCN3C(=O)[C@H](CC4=CN=CN4)NC(=O)[C@@H]5CCCN5C(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CCSC)NC(=O)CNC(=O)CNC(=O)CNC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCCNC(=N)N)N

QPMHUXBSHGAVGD-MCDIZDEASA-N

InChI=1S/C75H118N20O22S/c1-12-39(7)59(90-70(111)57(37(3)4)88-68(109)52-19-16-27-95(52)74(115)49(30-44-32-78-36-82-44)87-67(108)51-18-15-26-94(51)53(97)33-79-62(103)41(9)83-63(104)45(76)24-28-118-11)72(113)89-58(38(5)6)71(112)91-60(40(8)13-2)73(114)92-61(42(10)96)69(110)80-34-54(98)93-25-14-17-50(93)66(107)86-48(29-43-31-77-35-81-43)65(106)84-46(20-22-55(99)100)64(105)85-47(75(116)117)21-23-56(101)102/h31-32,35-42,45-52,57-61,96H,12-30,33-34,76H2,1-11H3,(H,77,81)(H,78,82)(H,79,103)(H,80,110)(H,83,104)(H,84,106)(H,85,105)(H,86,107)(H,87,108)(H,88,109)(H,89,113)(H,90,111)(H,91,112)(H,92,114)(H,99,100)(H,101,102)(H,116,117)/t39-,40-,41-,42+,45-,46-,47-,48-,49-,50-,51-,52-,57-,58-,59-,60-,61-/m0/s1

(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[2-[[(2S,3R)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-1-[2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]propanoyl]amino]acetyl]pyrrolidine-2-carbonyl]amino]-3-(1H-imidazol-5-yl)propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutanoyl]amino]-3-methylpentanoyl]amino]-3-methylbutanoyl]amino]-3-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]acetyl]pyrrolidine-2-carbonyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-carboxybutanoyl]amino]pentanedioic acid

NFAT Inhibitor; 249537-73-3; VIVIT peptide; NFAT Inhibitor trifluoroacetate salt; ...; VIVIT peptide;11R-VIVIT;ABM-7733;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~100 mg/mL (~27.99 mM)

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.)