PI3K

The PDGFR 740Y-P peptide stimulates a mitogenic response in muscle cells. The ability of the 740Y-P peptide to stimulate mitogenesis is highly specific and not a general feature of a cell permeable SH2 domain binding peptides[2].
Through the well-known PI 3-kinase-Akt survival cascade, 740Y-P is just as effective as a growth factor (FGF2) at promoting neuronal cell survival. The PDGFR740Y-P peptide does not require insulin and can stimulate neuronal cell survival[1].

740-Y-P increases the extent of AKT and PI3K phosphorylation in the Alzheimer's disease rat model and decreases the degree of ROS levels in the hippocampal tissues treated with A(25-32). Researchers found that GABAB receptor activation restored spatial memory and learning ability of AD rats and suppressed the neuronal apoptosis and hippocampal atrophy by activating the PI3K/Akt signaling pathway. Additionally, GABAB receptor activation reduced the oxidative stress injury by lowering the MDA levels and increased the SOD, GSH-Px, and CAT levels via activation of the PI3K/Akt signaling pathway. Conclusion: Taken together, the results suggest that GABAB receptor activation repressed the oxidative stress injury implicated in neurons in AD rats via PI3K/Akt signaling pathway activation which may suggest a potential new therapeutic target for AD.

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Moreover, the successfully modeled rats were treated with baclofen (the AD + baclofen group), 740-Y-P (PI3K/Akt signaling pathway agonist) (the AD + 740-Y-P group), and LY294002 (the AD + LY294002 group), respectively, with 10 rats in each group. The hippocampal extracts from rats were differently treated for detection of the extent of PI3k and Akt phosphorylation by western blot analysis (Fig. 3B, C). The extent of PI3k and Akt phosphorylation was declined in rats of the AD + baclofen group, the AD +740-Y-P group, the AD + LY294002 group, and the AD + baclofen + LY294002 group compared with the normal group. Moreover, compared with the AD group, the extent of PI3k and Akt phosphorylation was enhanced in the AD + baclofen group, and the AD + 740-Y-P group, however, it was reduced in the AD + LY294002 group. [3]
Furthermore, we analyzed the expression of Bax, Bcl-2, cleaved caspase 3, and Caspase-3 in the hippocampal extracts in each group using western blot analysis (Fig. 3B, D). It was revealed that the expression of Bcl-2 and Caspase-3 increased in the AD + baclofen group and the AD + 740-Y-P group along with the decreased expression of Bax and cleaved caspase-3 expression; however, in the AD + LY294002 group, Bcl-2 and Caspase-3 expression was remarkably reduced while the Bax and cleaved caspase-3 expression increased. The rats in the AD + baclofen + LY294002 group exhibited downregulated expression of Bcl-2 and Caspase-3 whereas the upregulated expression of Bax and cleaved caspase-3 was observed compared with the rats in the AD + baclofen group. Collectively, these results suggested that the GABAB receptor could activate the PI3K/Akt signaling pathway to suppress apoptosis of hippocampal cells. [3]

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In contrast to the Aβ-treated hippocampal tissues, the degree of ROS levels was decreased in the Aβ-treated hippocampal tissues with baclofen or 740-Y-P treatment. ELISA results showed that the normal group exhibited a significantly lower MDA level while distinctly higher levels of SOD, GSH-Px, and CAT than the AD group, the AD + baclofen group, the AD + 740-Y-P group, the AD + LY294002 group, and the AD + baclofen + LY294002 group (p < 0.05). The MDA level was notably lower while levels of SOD, GSH-Px, and CAT were significantly higher in the AD + baclofen or AD +740-Y-P group whereas in the AD + LY294002 group, the MDA level was remarkably elevated but the levels of SOD, GSH-Px, and CAT were reduced (all p < 0.05). [3]
The apoptosis of cultured hippocampal neurons was detected by flow cytometry with the combination of baclofen, 740-Y-P, LY294002, and Aβ. The results showed that (Fig. 6B), compared with the control group, the number of apoptotic cells in other groups was significantly increased, compared with the Aβ group, the apoptotic rate in the Aβ+ baclofen group and the Aβ+740-Y-P group was reduced, while the apoptotic rate in the Aβ+ LY294002 group was increased in contrast to the Aβ+ LY294002 group. [3]

The binding of small phosphopeptides to the SH2 domains of the p85 regulatory subunit of PI 3-kinase can activate the enzyme in vitro. In the present study a cell-permeable peptide that binds specifically to the SH2 domains of p85 has been evaluated for its ability to stimulate a mitogenic response in the C2 muscle cell line. This peptide, in contrast to four other SH2-binding peptides, was as effective as serum, EGF, and FGF at stimulating entry into S-phase. The response to the p85 binding peptide, but not FGF, was inhibited by wortmannin and rapamycin, indicating that the peptide activates the PI 3-kinase/S6 kinase signalling pathway. The peptide response was not inhibited by the MEK inhibitor (PD098059) and did not stimulate Erk phosphorylation. Thus, there would appear to be no direct cross-talk between the pathway activated by the p85 binding peptide and the p42/p44 MAPK cascade [2].

740 Y-P increases the extent of AKT and PI3K phosphorylation in the Alzheimer's disease rat model and decreases the degree of ROS levels in the hippocampal tissues treated with A(25-32).NIH HBSS with 10% FCS, Ca2+ and Mn2+ free, and either 50 g/ml of the 740-Y-P peptide or an equal volume of PBS as a control are incubated with 2 106 3T3 cells in suspension at 37°C. Cells are centrifuged, cleaned, and trypsinized after 2 hours to break down non-internalized peptide. Then, the cells are resuspended in lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 10% Glycerol, 2% NP40, 0.25% deoxycholate, 1 mM EDTA, 1 mM Vanadate, Protease Inhibitors Complete" Cocktail from Boehringer-Mannheim) and incubated at 4°C for 1 hour. Lysates are clarified by centrifuging at 1.4×104g for 5 min, and the supernatants are then incubated with streptavidin-agarose beads for 1h. After that, beads are washed three times in lysis buffer, boiled in SDS sample buffer, resolved by SDS-PAGE on a 12% gel, transferred to nitrocellulose, and immunoblotted with the p85 monoclonal antibody.

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Isolation and culture of hippocampal neuronal cells [3]
Hippocampus was isolated bluntly accompanied by vessels and meninges removed. Hippocampus was sectioned into pieces with a diameter of 0.4 mm. Sections were then reacted with 0.25% trypsin and 0.04 DNA enzyme for 12 min and the reaction was terminated by adding horse serum. Cells were dispersed by pipette trituration for 10 times. The cell suspension was cultured in a MEM containing 10% FBS, 5% horse serum, 25 mmol/L KCI, 10 mmol/L HEPES, 105 U/L penicillin and 0.1 g/L streptomycin. Following the filtration by a nylon mesh filter with a diameter of 75μm, samples were cultured in a 35 mm dish coated with Poly-L-lysine hydrobromide slides and incubated in 5% CO2 and 95% O2 at 37°C with a density of 0.6×109 L–1. At day 3, 5μmol/L cytarabine was supplemented to the culture and the solution was replaced after every 24 h. At day 7 of post culture, samples were subjected to neuron-specific enolase immunocytochemistry staining via the SP method to identify hippocampal neuronal cells. Cultured cells were treated with Aβ (with the final concentration of 25μmol/L), baclofen (with the final concentration of 25μmol/L), 740-Y-P (with the final concentration of 20μmol/L),or LY294002 (with the final concentration of 10μmol/L), alone or in combination. Each treatment lasted for 24 h. Untreated cells were taken as control.

To evaluate this hypothesis, a rat AD model was established by intraperitoneal injection of the GABAB receptor agonist (baclofen), PI3K/Akt signaling pathway agonist (740-Y-P), and antagonist (LY294002), respectively. The effects of GABAB activation on spatial memory and learning ability in the AD rats were measured by Morris water maze. Whereas the effects of GABAB and PI3K/Akt signaling pathway on apoptosis and oxidative stress injury were determined in vivo and in vitro using primary neuronal cultures [3].

Seventy healthy male adult SD rats [specific pathogen-free (SPF); age, 2– 3 months, weighed 32.10±24.70 g were housed with free access to water and food in a 12/12 h day/night cycle at 25±2°C. After seven days of acclimatization, a total of 60 rats were randomly grouped with 10 rats each group. AD modeled rats were intraperitoneally injected with GABAB receptor agonist baclofen (2.0 mg/kg), PI3K/Akt signaling pathway agonist 740-Y-P (10 mg/kg), or PI3K/Akt signaling pathway inhibitor LY294002 (20 mg/kg), or both baclofen (2.0 mg/kg) +LY294002 (20 mg/kg). Rats were treated differently for consecutive 6 weeks. The experimental protocol for treatments and behavioral tests can be seen in Fig. 1. [3]

[1]. Mol Cell Neurosci. 1999 Apr;13(4):272-80.

[2]. Biochem Biophys Res Commun. 1998 Oct 9;251(1):148-52.

[3]. J Alzheimers Dis. 2020;76(4):1513-1526.

PI3-kinase has become a key enzyme regulating neuronal cell survival. However, it remains unclear whether endogenous PI3-kinase activation regulated by the p85 subunit is sufficient to promote cell survival. Furthermore, it is also unclear whether FGF family growth factors promote cell survival through a PI3-kinase-dependent pathway. We previously developed a cell-permeable p85-binding peptide and demonstrated its ability to stimulate a mitogenic response in muscle cells dependent on the PI3-kinase/p70 S6 kinase pathway. In this study, we found that this peptide rescued serum deprivation-induced cerebellar granule cell death, and the response was comparable to that of growth factor (FGF2). Experiments using wortmannin, LY294002, and rapamycin showed that the peptide's survival-promoting response depended on PI3-kinase activity, rather than p70 S6 kinase activity. The peptide response was associated with PI3-kinase-dependent Akt phosphorylation, Akt being a known downstream effector molecule in the PI3-kinase survival cascade. In contrast to the survival response stimulated by p85-binding peptides, the response stimulated by FGF2 was not inhibited by wortmannin or LY294002 and was independent of Akt phosphorylation. Therefore, we can conclude that activation of the p85-regulated endogenous PI3-kinase pool is sufficient to maintain cell survival; however, growth factors such as FGF2 can obviously promote cell survival in a PI3-kinase-independent manner. [1]

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Small phosphopeptides can activate the enzyme in vitro by binding to the SH2 domain of the PI3-kinase p85 regulatory subunit. In this study, we evaluated a cell-penetrating peptide that specifically binds to the SH2 domain of p85 and examined its ability to stimulate mitosis in the C2 muscle cell line. Compared with four other SH2-binding peptides, this peptide was as effective as serum, EGF and FGF in stimulating cells to enter S phase. Wortmannin and rapamycin inhibited the cell proliferation response induced by p85-binding peptides (but not FGF), indicating that the peptide activated the PI3K/S6 kinase signaling pathway. The MEK inhibitor (PD098059) does not inhibit the induction response of this peptide, and the peptide does not stimulate Erk phosphorylation. Therefore, there appears to be no direct interaction between the p85-binding peptide-activated pathway and the p42/p44 MAPK cascade. [2]

C141H222N43O39PS3.XC2HF3O2

3270.70 (free acid)

1236188-16-1

Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Ser-Asp-Gly-Gly-{Tyr(PO2)}-Met-Asp-Met-Ser

RQIKIWFQNRRMKWKKSDGG-{PO2Y}-MDMS

Typically exists as solid at room temperature

(3S)-3-[[(2S)-2-[[(2S)-2-[[2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-6-amino-2-[[(2S,3S)-2-[[(2S)-5-amino-2-[[(2S)-2-amino-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]amino]hexanoyl]amino]-3-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-3-phenylpropanoyl]amino]-5-oxopentanoyl]amino]-4-oxobutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-carbamimidamidopentanoyl]amino]-4-methylsulfanylbutanoyl]amino]hexanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]hexanoyl]amino]hexanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]acetyl]amino]acetyl]amino]-3-(4-phosphonooxyphenyl)propanoyl]amino]-4-methylsulfanylbutanoyl]amino]-4-[[(2S)-1-[[(1S)-1-carboxy-2-hydroxyethyl]amino]-4-methylsulfanyl-1-oxobutan-2-yl]amino]-4-oxobutanoic acid TFA salt

740 Y-P TFA; 740 YP; 740YPDGFR TFA; PDGFR740Y-P TFA; 740 Y P; 740-YPDGFR; PDGFR 740 Y-P; 740 YPDGFR; PDGFR 740Y-P; H-Arg-Gln-Ile-Lys-Ile-Trp-Phe-Gln-Asn-Arg-Arg-Met-Lys-Trp-Lys-Lys-Ser-Asp-Gly-Gly-Tyr(PO3H2)-Met-Asp-Met-Ser-OH; Alternative Name: PDGFR740Y-P; 740 Y-P?; PDGFR 740Y-P

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)

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

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.)