The N-terminal active site of Angiotensin I-Converting Enzyme (ACE) is identified as the primary and specific site for the hydrolysis of N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) (Km = 31 µM, kcat = 16 s⁻¹, kcat/Km = 0.5 µM⁻¹·s⁻¹). The C-terminal active site of ACE also hydrolyzes AcSDKP but with a much lower catalytic efficiency (Km = 39 µM, kcat = 0.40 s⁻¹, kcat/Km = 0.01 µM⁻¹·s⁻¹). [1]
TGFβ1/Smad2 signaling pathway (inhibition of Smad2 phosphorylation and nuclear translocation). [2]

N-Acetyl-Ser-Asp-Lys-Pro is particularly destroyed by ACE, and its plasma levels increase considerably with ACE inhibitor treatment. Flow cytometry of rat cardiac fibroblasts treated with N-Acetyl-Ser-Asp-Lys-Pro indicated that cell progression from the G0/G1 phase of the cell cycle to the S phase was significantly slowed. Furthermore, Smad2 phosphorylation and nuclear translocation were decreased in cardiac fibroblasts treated with N-Acetyl-Ser-Asp-Lys-Pro [1]. N-acetyl-seryl-aspartyl-lysyl-proline appears to exert this function by inhibiting the effects of stem cell-specific proliferation stimulators and acting preferentially on quiescent progenitor cells [2] . N-Acetyl-Ser-Asp-Lys-Pro suppresses collagenase expression, and activation is related with increased production of TIMP-1 and TIMP-2. N-Acetyl-Ser-Asp-Lys-Pro normalizes IL-1β-mediated increases in MMP-2 and MMP-9 activity and MMP-13 expression [3].

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Kinetic studies using recombinant human ACE enzymes (wild-type, N-functional domain mutant, C-functional domain mutant) demonstrated that AcSDKP is hydrolyzed by both the N- and C-terminal active sites of ACE. However, the catalytic efficiency (kcat/Km) of the N-terminal active site is approximately 50-fold higher than that of the C-terminal active site, establishing it as the predominant site for AcSDKP cleavage. [1]
The hydrolysis of AcSDKP by ACE is dependent on pH and chloride ions. Optimal activity for all three enzyme forms (wild-type, N- and C-domain mutants) occurs at pH 6.5. The activities of wild-type ACE and the N-domain mutant are optimal at ~50 mM NaCl and are inhibited at higher chloride concentrations, while the C-domain mutant achieves optimal activity at ~300 mM NaCl. Phosphate ions strongly inhibit AcSDKP hydrolysis by all enzyme forms. [1]
A monoclonal antibody (i2H4) specifically directed against the N-terminal active site of ACE completely inhibited AcSDKP hydrolysis by the N-domain mutant and wild-type ACE, but had minimal effect on hydrolysis by the C-domain mutant, confirming the specificity of AcSDKP for the N-terminal site. [1]
ACE inhibitors captopril and lisinopril inhibited AcSDKP hydrolysis. For wild-type ACE and the N-domain mutant, IC₅₀ values for both inhibitors were approximately 2 nM. For the C-domain mutant, IC₅₀ values were about 30 nM, indicating different inhibitory potencies against the two active sites. [1]
AcSDKP (0.01 and 1 nmol/L) significantly inhibited serum-stimulated proliferation of neonatal rat cardiac fibroblasts, as measured by MTS assay (absorbance reduced from 0.445±0.009 to 0.379±0.009 and 0.371±0.005, respectively; P<0.001).
AcSDKP (1 nmol/L) inhibited TGFβ1-stimulated proliferation of neonatal rat cardiac fibroblasts (absorbance reduced from 0.393±0.009 to 0.358±0.012).
AcSDKP (0.01 and 1 nmol/L) inhibited the transition of cardiac fibroblasts from G0/G1 phase to S phase, as shown by flow cytometry (G0/G1 phase cells remained at 82.4±0.4% and 83.7±0.5% vs. 75±1.4% in serum-stimulated control).
AcSDKP (1 nmol/L) decreased TGFβ1-induced Smad-binding element (SBE) promoter activity by 55.3±9.7% in cardiac fibroblasts transfected with a Smad-sensitive luciferase reporter construct. [2]
AcSDKP decreased TGFβ1-stimulated phosphorylation of Smad2 in a dose-dependent manner in cardiac fibroblasts, as shown by Western blot. [2]
AcSDKP (1 and 100 nmol/L) decreased nuclear translocation of Smad2 in TGFβ1-stimulated cardiac fibroblasts (nuclear staining positive cells decreased to 58±3.4% and 56±4.04% vs. 72±4.9% in TGFβ1-only group). [2]
AcSDKP (1 nmol/L) stimulated the proliferation of rat aortic smooth muscle cells (PAC1 cells) in the presence of 5% FBS (cell number increased from 42,375±7,368 to 79,240±3,488 cells/mL). [2]

N-Acetyl-Ser-Asp-Lys-Pro inhibits phenylephrine down-regulation, albuminuria, collagen deposition, and inflammatory cell infiltration that are brought on by hypertension. In hypertensive mice, this may have renoprotective properties [4].

The standard enzymatic assay for AcSDKP hydrolysis was performed in triplicate at 37°C in a buffer containing Tris-maleate, ZnSO₄, and NaCl. Enzymes (wild-type or mutant recombinant ACE) were incubated with a concentration range of unlabeled AcSDKP spiked with a trace amount of tritium-labeled AcSDKP ([³H]AcSDKP). The reaction was stopped by freezing. Carrier peptides were added, and the mixture was subjected to high-voltage paper electrophoresis to separate the substrate from the radioactive dipeptide product ([³H]Lys-Pro). Radioactivity associated with each spot was quantified by scintillation counting to calculate the percentage of hydrolysis and initial velocity. [1]
To study pH dependence, enzymes were incubated with AcSDKP in buffers of varying pH (5.0-9.0). Activity was measured after a fixed incubation time. [1]
To study chloride activation, enzymes were incubated with AcSDKP in buffers containing a range of NaCl concentrations (0-600 mM), and activity was determined. [1]
For monoclonal antibody inhibition, enzymes were pre-incubated overnight at 4°C with varying molar ratios of the anti-N-domain antibody i2H4. Residual enzymatic activity towards AcSDKP was then measured. [1]
For ACE inhibitor studies, enzymes were pre-incubated with varying concentrations of captopril or lisinopril for 30 minutes at 37°C. Residual activity towards AcSDKP was then measured. For comparison, inhibition of the hydrolysis of another substrate, Hip-His-Leu (a substrate primarily cleaved by the C-terminal active site), was also assessed under similar pre-incubation conditions. [1]

Cell proliferation assay (MTS assay): Cardiac fibroblasts were plated in 96-well plates, synchronized in low-serum medium (0.1% FBS) for 24 hours, then stimulated with 10% FBS or TGFβ1 (1 ng/mL) with or without AcSDKP. After 24 hours, MTS reagent was added and incubated for 4 hours, and absorbance was measured at 490 nm. [2]
Cell cycle analysis (flow cytometry): Cardiac fibroblasts were treated with or without AcSDKP, detached with trypsin, washed, incubated with propidium iodide, and analyzed by flow cytometry to determine cell cycle distribution. [2]
Luciferase reporter assay: Cardiac fibroblasts were transfected with a Smad-sensitive luciferase reporter plasmid (pGL3ti(SBE)4) and a control plasmid (pRL-CMV). After 24 hours, cells were treated with TGFβ1 (5 ng/mL) with or without AcSDKP (added 15 minutes before TGFβ1). Luciferase activity was measured 24 hours later using a dual-luciferase assay system. [2]
Western blot analysis: Cardiac fibroblasts were treated with TGFβ1 and AcSDKP. Whole cell lysates were prepared after 24 hours, and proteins (Smad2, phospho-Smad2, Smad4, Smad7, GAPDH) were detected using specific antibodies and enhanced chemiluminescence. [2]
Immunocytochemistry: Cardiac fibroblasts were fixed, permeabilized, blocked, and incubated with Smad2 antibody overnight at 4°C. After washing, cells were incubated with HRP-conjugated secondary antibody and streptavidin-HRP, followed by DAB staining to visualize nuclear Smad2. [2]
Smooth muscle cell proliferation assay (Coulter counter): PAC1 cells were serum-starved for 24 hours, then stimulated with 5% FBS with or without AcSDKP. Cells were counted 24 hours later using a Coulter counter. [2]

AcSDKP is an endogenous tetrapeptide that is specifically degraded by angiotensin-converting enzyme (ACE). During ACE inhibitor treatment, AcSDKP plasma levels can rise to 20 nmol/L. [2]

[1]. The hemoregulatory peptide N-acetyl-Ser-Asp-Lys-Pro is a natural and specificsubstrate of the N-terminal active site of human angiotensin-converting enzyme. J Biol Chem. 1995 Feb 24;270(8):3656-61.

[2]. N-acetyl-Ser-Asp-Lys-Pro inhibits phosphorylation of Smad2 in cardiac fibroblasts. Hypertension. 2002 Aug;40(2):155-61.

[3]. N-acetyl-Ser-Asp-Lys-Pro inhibits interleukin-1β-mediated matrix metalloproteinase activation in cardiac fibroblasts. Pflugers Arch. 2013 Oct;465(10):1487-95.

[4]. Renal protective effects of N-acetyl-Ser-Asp-Lys-Pro in deoxycorticosterone acetate-salt hypertensive mice. J Hypertens. 2011 Feb;29(2):330-8.

Golatriide is a tetrapeptide with the sequence Ser-Asp-Lys-Pro, where an acetyl group is attached to the N-terminal amino group. It selectively inhibits the proliferation of primitive hematopoietic cells and has anti-inflammatory, anti-fibrotic and angiogenic properties. It can act as an anti-inflammatory and angiogenic agent. Its function is related to Ser-Asp-Lys-Pro. It is the conjugate acid of goratide (1-).

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N-acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a tetrapeptide that was first isolated from fetal bovine bone marrow. It plays a negative regulatory role in the proliferation of hematopoietic stem cells (blood regulatory peptide) by preventing hematopoietic stem cells from entering the S phase of the cell cycle. [1]
This study found that AcSDKP is the first known natural and highly specific substrate of the N-terminal active site of ACE with a kinetic constant within the physiological range. This suggests that ACE may participate in the regulation of local AcSDKP concentration in vivo through its N-terminal domain. [1]
The authors propose that specific inhibitors of the N-terminal active site of ACE may be used to improve the in vivo stability and physiological concentration of AcSDKP. This may be beneficial for cancer treatment by reversibly maintaining normal hematopoietic stem cells in a quiescent state, thereby protecting these cells during cytotoxic chemotherapy without interfering with the blood pressure regulation function of the C-terminal active site of ACE. [1]
AcSDKP is a specific substrate of the N-terminal active site of ACE and is inactivated by ACE. [2]
During ACE inhibitor treatment, AcSDKP levels were significantly elevated, suggesting that ACE inhibitors may inhibit cardiac fibrosis through a novel pathway (independent of angiotensin II). [2]
AcSDKP inhibits cardiac fibroblast proliferation but stimulates vascular smooth muscle cell proliferation, indicating that it has cell type-specific effects. [2]

C20H33N5O9

487.50412

487.227

127103-11-1

N-Acetyl-Ser-Asp-Lys-Pro acetate;N-Acetyl-Ser-Asp-Lys-Pro TFA

65938

Typically exists as solid at room temperature

1.4±0.1 g/cm3

992.0±65.0 °C at 760 mmHg

553.7±34.3 °C

0.0±0.6 mmHg at 25°C

1.565

-1.91

7

10

14

34

776

4

CC(NC(C(NC(C(NC(C(N1CCCC1C(O)=O)=O)CCCCN)=O)CC(O)=O)=O)CO)=O

HJDRXEQUFWLOGJ-AJNGGQMLSA-N

InChI=1S/C20H33N5O9/c1-11(27)22-14(10-26)18(31)24-13(9-16(28)29)17(30)23-12(5-2-3-7-21)19(32)25-8-4-6-15(25)20(33)34/h12-15,26H,2-10,21H2,1H3,(H,22,27)(H,23,30)(H,24,31)(H,28,29)(H,33,34)/t12-,13-,14-,15-/m0/s1

(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-acetamido-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-6-aminohexanoyl]pyrrolidine-2-carboxylic acid

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.)