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.

PI 3-kinase has emerged as a key enzyme for regulating neuronal cell survival. However, it has not as yet been demonstrated whether activation of the endogenous pool of the enzyme, that is regulated by the p85 subunit, is sufficient to promote a survival response. It is also not known whether the FGF family of growth factors promote survival via a PI 3-kinase-dependent pathway. We have previously developed a cell permeable p85 binding peptide and shown that it can stimulate a mitogenic response in muscle cells that is dependent on a PI 3-kinase/p70 S6 kinase pathway. In the present study we show that this peptide can rescue cerebellar granule cells from death induced by serum deprivation and that this response is comparable to a growth factor response (FGF2). Experiments with wortmannin, LY294002, and rapamycin suggest that the peptide survival response is dependent on PI 3-kinase activity, but not p70 S6 kinase activity. The peptide response was correlated with a PI 3-kinase-dependent phosphorylation of Akt, an established downstream effector in the PI 3-kinase survival cascade. In contrast to the survival response stimulated by the p85 binding peptide, the response stimulated by FGF2 was not inhibited by wortmannin or LY294002, nor was it associated with phosphorylation of Akt. Thus we can conclude that activation of the endogenous pool of PI 3-kinase that is regulated by p85 is sufficient for cell survival; however, growth factors such as FGF2 can clearly support survival in a PI 3-kinase-independent manner.[1]

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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]

C141H222N43O39PS3

3270.7049

3268.561

1236188-16-1

740 Y-P TFA

90488730

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

R-Q-I-K-I-W-F-Q-N-R-R-M-K-W-K-K-S-D-G-G-{PO2-Y}-M-D-M-S

White to off-white solid

1.5±0.1 g/cm3

1.679

-6.32

50

50

117

227

7280

25

S(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])C([H])([H])C(N([H])C([H])([H])C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(N([H])[C@]([H])(C(=O)O[H])C([H])([H])O[H])=O)C([H])([H])C([H])([H])SC([H])([H])[H])=O)C([H])([H])C(=O)O[H])=O)C([H])([H])C([H])([H])SC([H])([H])[H])=O)C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])OP(=O)(O[H])O[H])=O)=O)=O)C([H])([H])C(=O)O[H])=O)C([H])([H])O[H])=O)C([H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H])=O)C([H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H])=O)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12)=O)C([H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H])=O)N([H])C([C@]([H])(C([H])([H])C([H])([H])C([H])([H])N([H])/C(=N/[H])/N([H])[H])N([H])C([C@]([H])(C([H])([H])C([H])([H])C([H])([H])N([H])/C(=N/[H])/N([H])[H])N([H])C([C@]([H])(C([H])([H])C(N([H])[H])=O)N([H])C([C@]([H])(C([H])([H])C([H])([H])C(N([H])[H])=O)N([H])C([C@]([H])(C([H])([H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C([C@]([H])(C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12)N([H])C([C@]([H])([C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])N([H])C([C@]([H])(C([H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])[H])N([H])C([C@]([H])([C@@]([H])(C([H])([H])[H])C([H])([H])C([H])([H])[H])N([H])C([C@]([H])(C([H])([H])C([H])([H])C(N([H])[H])=O)N([H])C([C@]([H])(C([H])([H])C([H])([H])C([H])([H])N([H])/C(=N/[H])/N([H])[H])N([H])[H])=O)=O)=O)=O)=O)=O)=O)=O)=O)=O)=O

XCGMILZGRGEWHL-QYGSNONCSA-N

InChI=1S/C141H222N43O39PS3/c1-8-75(3)114(184-128(208)94(46-48-108(148)188)164-116(196)84(146)32-25-56-156-139(150)151)136(216)174-90(38-20-24-55-145)127(207)183-115(76(4)9-2)137(217)180-101(65-80-70-160-86-34-16-14-31-83(80)86)132(212)175-99(62-77-28-11-10-12-29-77)130(210)170-93(45-47-107(147)187)123(203)177-102(66-109(149)189)133(213)169-92(40-27-58-158-141(154)155)119(199)167-91(39-26-57-157-140(152)153)120(200)171-95(49-59-225-5)124(204)166-88(36-18-22-53-143)121(201)176-100(64-79-69-159-85-33-15-13-30-82(79)85)131(211)168-87(35-17-21-52-142)118(198)165-89(37-19-23-54-144)122(202)181-105(73-185)135(215)178-103(67-112(192)193)117(197)162-71-110(190)161-72-111(191)163-98(63-78-41-43-81(44-42-78)223-224(220,221)222)129(209)172-96(50-60-226-6)125(205)179-104(68-113(194)195)134(214)173-97(51-61-227-7)126(206)182-106(74-186)138(218)219/h10-16,28-31,33-34,41-44,69-70,75-76,84,87-106,114-115,159-160,185-186H,8-9,17-27,32,35-40,45-68,71-74,142-146H2,1-7H3,(H2,147,187)(H2,148,188)(H2,149,189)(H,161,190)(H,162,197)(H,163,191)(H,164,196)(H,165,198)(H,166,204)(H,167,199)(H,168,211)(H,169,213)(H,170,210)(H,171,200)(H,172,209)(H,173,214)(H,174,216)(H,175,212)(H,176,201)(H,177,203)(H,178,215)(H,179,205)(H,180,217)(H,181,202)(H,182,206)(H,183,207)(H,184,208)(H,192,193)(H,194,195)(H,218,219)(H4,150,151,156)(H4,152,153,157)(H4,154,155,158)(H2,220,221,222)/t75-,76-,84-,87-,88-,89-,90-,91-,92-,93-,94-,95-,96-,97-,98-,99-,100-,101-,102-,103-,104-,105-,106-,114-,115-/m0/s1

(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

740 Y-P; 740 YP; 740YPDGFR; PDGFR740Y-P;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

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO: 25~100 mg/mL (7.6~30.6 mM)
Ethanol: 8 mg/mL (~2.5 mM)
H2O: 5 mg/mL (1.5 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (0.76 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 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (0.76 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (0.76 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)