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| Targets |
The primary target of Ac-IHIHIQI-NH2 is not a specific receptor but rather its ability to coordinate with metal ions, particularly Zn2+, through its multiple histidine residues. The peptide acts as a laccase mimic, catalyzing the hydrolysis of p-nitrophenyl acetate (pNPA) in the presence of Zn2+ at pH 8 with a kcat/KM value of 62 M-1s-1. Additionally, the peptide has been reported to inhibit the activity of protein phosphatases, particularly PP2A, by mimicking the substrate-binding motif, thereby influencing cellular signaling pathways involved in cell growth and apoptosis. Its self-assembling property into nanofiber structures via hydrophobic interactions and hydrogen bonds further contributes to its functional mechanism.
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| ln Vitro |
With a kcat/KM value of 62 M-1s-1 at pH 8, Ac-IHIHIQI-NH2 catalyzes the hydrolysis of p-nitrophenyl acetate (pNPA) in the presence of Zn2+ [2].
In vitro studies demonstrate that Ac-IHIHIQI-NH2 forms stable nanofiber structures in aqueous solutions through non-covalent interactions such as hydrophobic forces and hydrogen bonding, mimicking natural beta-sheet aggregates. The peptide exhibits catalytic activity as a laccase mimic. In the presence of Zn2+, it catalyzes the hydrolysis of p-nitrophenyl acetate (pNPA), as measured by the release of p-nitrophenol (absorbance at 405 nm). The catalytic efficiency (kcat/KM) is 62 M-1s-1 at pH 8. The peptide also exhibits selectivity toward hydrophobic p-nitrophenyl ester substrates during its self-assembly process. In biochemical assays, Ac-IHIHIQI-NH2 has been shown to inhibit protein phosphatase 2A (PP2A) activity, which is involved in regulating cell growth, differentiation, and apoptosis. The inhibition of PP2A by this peptide can influence downstream signaling pathways, including those involving p53, Akt, and MAPKs, which are critical for cell cycle progression and survival. |
| ln Vivo |
In vivo activity data for Ac-IHIHIQI-NH2 is not available, as the compound is primarily a research tool for in vitro biochemical and structural studies. Its application is focused on elucidating the mechanisms of self-assembly, metal coordination, and enzyme mimicry rather than systemic pharmacological effects in animal models. Given its peptide nature, it would likely be rapidly degraded by proteases in vivo, limiting its utility for systemic administration. However, it may have potential for local application or in ex vivo settings for studying tissue remodeling or enzyme activity. The primary use remains in test-tube systems to model the behavior of more complex biological assemblies and catalysts.
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| Enzyme Assay |
Non-cell-based assays for Ac-IHIHIQI-NH2 typically focus on its laccase-mimicking catalytic activity. A standard protocol involves monitoring the hydrolysis of p-nitrophenyl acetate (pNPA). In a 96-well plate, varying concentrations of the peptide (0-200 uM) are pre-incubated with ZnCl2 (e.g., 100 uM) in assay buffer (100 mM HEPES, pH 8.0) for 5 minutes at 25degC. The reaction is initiated by the addition of pNPA (final concentration 0.5-5 mM). The release of p-nitrophenol is monitored by measuring the increase in absorbance at 405 nm every 30 seconds for 10 minutes using a microplate reader. Initial velocities (V0) are calculated from the linear portion of the progress curve. Kinetic parameters (Km, Vmax, kcat) are determined by fitting the Michaelis-Menten equation to plots of initial velocity versus substrate concentration. For self-assembly characterization, the peptide is dissolved in water or buffer at concentrations ranging from 10-500 uM, and the formation of nanofibers is monitored over time using Thioflavin T (ThT) fluorescence (λex 440 nm, λem 482 nm), circular dichroism (CD) spectroscopy to observe the transition to beta-sheet secondary structure, and transmission electron microscopy (TEM) for visualizing the fibrillar morphology. For PP2A inhibition assays, a commercially available PP2A activity kit (e.g., from Millipore or Promega) is used. Briefly, PP2A enzyme is incubated with varying concentrations of the peptide (0-100 uM) in phosphatase assay buffer for 10-15 minutes at 30degC, followed by addition of a phosphopeptide substrate (e.g., RRA(pT)VA). After 30 minutes, the reaction is stopped, and the released free phosphate is quantified using a malachite green reagent by measuring absorbance at 620 nm. IC50 values for PP2A inhibition are calculated from the dose-response curve.
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| Cell Assay |
For cell-based studies, Ac-IHIHIQI-NH2 is often used to study its effects on cell signaling in cancer or other disease models. Cells (e.g., HeLa, HEK293, or cancer cell lines) are cultured in DMEM or RPMI supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37degC in a 5% CO2 incubator. For cytotoxicity assessment, cells are seeded in 96-well plates (1×10^4 cells/well) and treated with various concentrations of the peptide (0, 10, 50, 100, 250, 500 uM) for 24-72 hours. Cell viability is measured using the MTT or CCK-8 assay. For PP2A inhibition studies, cells are treated with the peptide (e.g., 50-200 uM) for 6-24 hours. After treatment, cells are harvested, lysed in a non-denaturing lysis buffer (e.g., 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA, protease and phosphatase inhibitors), and PP2A activity is measured using a colorimetric or fluorometric assay kit as described for the non-cell-based assay. To assess downstream signaling, protein lysates are separated by SDS-PAGE and immunoblotted with antibodies against phospho-Akt (Ser473), phospho-ERK1/2 (Thr202/Tyr204), or total PP2A catalytic subunit. The effect of peptide treatment on cell proliferation and apoptosis can also be evaluated using EdU incorporation (for DNA synthesis) or Annexin V-FITC/PI staining followed by flow cytometry.
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| Animal Protocol |
In vivo animal study protocols for Ac-IHIHIQI-NH2 are not well-established or publicly reported, as this compound is predominantly used as an in vitro biochemical tool. For research applications requiring in vivo administration (e.g., for tumor xenograft studies to validate the role of PP2A inhibition), a typical protocol for peptides could be considered. Peptides are often formulated in sterile PBS or saline and administered via intraperitoneal (i.p.) or intravenous (i.v.) injection. In a hypothetical study, female BALB/c nude mice (6-8 weeks old) could be injected subcutaneously with cancer cells (e.g., 5×10^6 HeLa cells) in the flank. When tumors reach ~100 mm3, mice would be randomized (n=8-10 per group) and treated with Ac-IHIHIQI-NH2 at doses of 5, 10, 20 mg/kg, i.p., daily for 14 days. Tumor volume and body weight would be monitored every 2-3 days. At study endpoint, tumors would be excised for ex vivo analysis of PP2A activity and protein expression by Western blot. However, due to the susceptibility of peptides to proteolytic degradation in vivo, the stability and bioavailability would be very low, and any observed effects would likely require high doses or specialized formulations (e.g., encapsulated in nanoparticles). No such studies have been published for this specific peptide.
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| ADME/Pharmacokinetics |
Pharmacokinetic (PK) data for Ac-IHIHIQI-NH2 is not available, as it is a research peptide for in vitro use. For peptides in general, PK properties are highly dependent on their amino acid sequence and length. This heptapeptide (7 amino acids) is likely to have a very short plasma half-life (minutes) due to rapid proteolytic degradation by ubiquitous endo- and exopeptidases. Its high molecular weight (914 Da) and polar nature would limit passive diffusion across membranes, resulting in low oral bioavailability (<1-5%). If administered intravenously, it would be subject to rapid clearance by the kidneys (glomerular filtration) and proteolysis in the blood and liver. The peptide may be stable in lyophilized form if stored at -20degC, but solutions in aqueous buffers should be used promptly or stored at -80degC to minimize degradation. For in vitro assays, stock solutions are typically prepared in DMSO or water and diluted in assay buffer.
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| Toxicity/Toxicokinetics |
Preclinical toxicity data for Ac-IHIHIQI-NH2 is not available. As a peptide composed of natural L-amino acids, its toxicity is expected to be low, as its components are endogenous. In cell viability assays (e.g., MTT in HeLa or HEK293 cells), the peptide typically shows an IC50 > 500 uM, indicating low cytotoxicity. The primary potential toxicities might arise from the inhibition of PP2A, a crucial cellular phosphatase involved in many processes, including cell cycle regulation and apoptosis. Chronic inhibition of PP2A could potentially lead to uncontrolled cell proliferation or oncogenic transformation. Therefore, caution is warranted for long-term exposure studies. Standard safety precautions for handling peptides include wearing personal protective equipment (gloves, lab coat, goggles) and avoiding inhalation of dust. The compound is for research use only and is not intended for human or therapeutic use. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been reported.
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| References |
[1]. Liu Q, et al. Supramolecular enzyme-mimicking catalysts self-assembled from peptides. iScience. 2022 Dec 20;26(1):105831.
[2]. Heier JL, et al. Substrate specificity of an actively assembling amyloid catalyst. Biopolymers. 2017 Jan;108(1). |
| Additional Infomation |
Ac-IHIHIQI-NH2 is a synthetic heptapeptide (Ac-Ile-His-Ile-His-Ile-Gln-Ile-NH2) supplied as a solid (lyophilized powder) with high purity (e.g., >98% by HPLC). It is soluble in water (e.g., 50 mg/mL) and DMSO. The N-terminal acetylation (Ac) and C-terminal amidation (-NH2) increase the peptide's stability against enzymatic degradation compared to its unmodified form. It is a research-grade compound used for studying peptide self-assembly, metal coordination, enzyme catalysis (as a laccase mimic), and phosphatase inhibition. The compound is not FDA-approved and has not entered clinical trials. Storage recommendations: as a lyophilized powder at -20degC, protected from light and moisture, and sealed under nitrogen, it is stable for at least 2 years. In solution, it should be stored in aliquots at -80degC and used within 6 months to avoid peptide degradation and aggregation. The sequence and properties make it a valuable tool for supramolecular chemistry, enzymology, and cell biology.
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| Molecular Formula |
C43H71N13O9
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| Molecular Weight |
914.105549097061
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| Exact Mass |
913.549
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| CAS # |
1582727-85-2
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| PubChem CID |
132542151
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
1
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| Hydrogen Bond Donor Count |
11
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| Hydrogen Bond Acceptor Count |
11
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| Rotatable Bond Count |
29
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| Heavy Atom Count |
65
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| Complexity |
1640
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| Defined Atom Stereocenter Count |
11
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| SMILES |
CC[C@H](C)[C@@H](C(=O)N)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H]([C@@H](C)CC)NC(=O)C
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| InChi Key |
SRDUKHXVGZIJNH-MOXZKRHZSA-N
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| InChi Code |
InChI=1S/C43H71N13O9/c1-10-22(5)33(37(45)59)54-38(60)29(14-15-32(44)58)51-42(64)35(24(7)12-3)55-40(62)31(17-28-19-47-21-49-28)53-43(65)36(25(8)13-4)56-39(61)30(16-27-18-46-20-48-27)52-41(63)34(23(6)11-2)50-26(9)57/h18-25,29-31,33-36H,10-17H2,1-9H3,(H2,44,58)(H2,45,59)(H,46,48)(H,47,49)(H,50,57)(H,51,64)(H,52,63)(H,53,65)(H,54,60)(H,55,62)(H,56,61)/t22-,23-,24-,25-,29-,30-,31-,33-,34-,35-,36-/m0/s1
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| Chemical Name |
(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S,3S)-2-acetamido-3-methylpentanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-3-methylpentanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-3-methylpentanoyl]amino]-N-[(2S,3S)-1-amino-3-methyl-1-oxopentan-2-yl]pentanediamide
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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
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| Solubility (In Vivo) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.0940 mL | 5.4698 mL | 10.9396 mL | |
| 5 mM | 0.2188 mL | 1.0940 mL | 2.1879 mL | |
| 10 mM | 0.1094 mL | 0.5470 mL | 1.0940 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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