CaM-kinase II (CaM-kinase II) [IC50 = 40 nM for CaM-kinase II activity;
Ki values: 18 nM (noncompetitive with respect to syntide-2), 320 nM (competitive with respect to autocamtide-2), 10 nM (competitive with respect to CaMK-(281-289));
IC50 for autophosphorylation of CaM-kinase II = 100 nM;
Inhibition of the constitutively active 30-kDa proteolytic fragment of CaM-kinase II: lowest Ki reported = 2-8 nM] [1]
Autocamtide-2-Related Inhibitory Peptide targets Ca2+/calmodulin-dependent protein kinase II (CaMKII). It is a highly selective inhibitor of this kinase, with an IC50 of 40 nM. The peptide inhibits CaMKII activity effectively regardless of the presence of Ca2+/calmodulin, unlike many other CaMKII inhibitors that require calmodulin for activity. Its selectivity for CaMKII over other kinases, such as PKC, makes it a specific probe for studying CaMKII function.

CaM kinase II was discovered to be effectively inhibited by autocamtide-2-related inhibitory peptide (ATP), a unique synthetic peptide that was active both in the presence and absence of Ca2+/calmodulin. Its IC50 was reported to be 40 nM. It has 500 and 50 times more potency than KN-93 and CaMK-(281-302Ala286), respectively, under the assay conditions employed [1].

---

AIP (40 nM IC50) inhibits CaM-kinase II activity in a concentration-dependent manner, being 50-fold more potent than CaMK-(281-302Ala286) (IC50 = 2 μM) and 500-fold more potent than KN-93 (IC50 = 20 μM). [1]
AIP at 10 μM does not significantly affect the activities of PKA, PKC, or CaM-kinase IV, whereas CaMK-(281-302Ala286) at 30 μM inhibits PKC and CaM-kinase IV. [1]
AIP potently inhibits autophosphorylation of CaM-kinase II with an IC50 of 100 nM. [1]
Kinetic analysis shows that AIP inhibition of CaM-kinase II is noncompetitive with respect to syntide-2 (Ki = 18 nM) and competitive with respect to autocamtide-2 (Ki = 320 nM) and CaMK-(281-289) (Ki = 10 nM). [1]
AIP (1 μM) completely inhibits CaM-kinase II activity regardless of calmodulin concentration (0.01-10 μM), whereas KN-93 inhibition is calmodulin-dependent and decreases with increasing calmodulin. [1]
AIP (1 μM) does not affect endogenous protein phosphorylation in CaM-kinase II-depleted rat brain extracts ("P-through" and "CaM-through" preparations) in the presence or absence of Ca2+, but markedly inhibits phosphorylation induced by exogenously added purified CaM-kinase II. [1]
In vitro, Autocamtide-2-Related Inhibitory Peptide effectively inhibits CaM kinase II with an IC50 of 40 nM. It is active in both the presence and absence of Ca2+/calmodulin. The peptide's potent and specific inhibition of CaMKII has been demonstrated in various in vitro kinase assays. Its selectivity over other kinases, including PKC, confirms its utility as a specific tool to dissect CaMKII-mediated signaling pathways.

Specific in vivo activity data for Autocamtide-2-Related Inhibitory Peptide are not extensively detailed in the provided search results. As a highly specific inhibitor, its primary use is in vitro to dissect CaMKII function. For in vivo applications, the peptide would face challenges such as poor cell permeability and rapid degradation. However, its potent in vitro activity makes it a valuable tool for studying the role of CaMKII in various cellular processes, and it could be used in cellular assays to confirm the involvement of CaMKII in specific signaling pathways.

CaM-kinase II activity was measured in a reaction mixture containing 40 mM Hepes-NaOH (pH 8.0), 5 mM magnesium acetate, 50 μM [γ-32P]ATP, 0.01% Tween 20, 100 μM syntide-2 as substrate, 0.3 mM CaCl2, 1 μM calmodulin, and purified CaM-kinase II from rat cerebral cortex. The reaction was carried out at 30°C, and activity was determined by the phosphocellulose paper method. [1]
The activity of the active 30-kDa proteolytic fragment of CaM-kinase II was measured similarly in the presence or absence of Ca2+/calmodulin as described previously. [1]
CaM-kinase IV activity was determined in the presence of 0.2 mM CaCl2 with 100 μM syntide-2 as substrate, using 40 mM Hepes-NaOH (pH 8.0) instead of 50 mM Hepes-NaOH (pH 7.0), and other conditions as previously described. [1]
PKA activity was assayed by phosphate incorporation into syntide-2 in a mixture containing 40 mM Mes-NaOH (pH 7.0), 5 mM magnesium acetate, 50 μM [γ-32P]ATP, 0.01% Tween 20, 100 μM syntide-2, and the catalytic subunit of PKA from bovine heart. The reaction was initiated by addition of the catalytic subunit and activity determined by the phosphocellulose paper method. [1]
PKC activity was measured in a mixture containing 40 mM Hepes-NaOH (pH 8.0), 10 mM magnesium acetate, 50 μM [γ-32P]ATP, 0.1 mM EGTA, 0.35 mM CaCl2, 0.01% Tween 20, 2.0 μg/ml 1,2-dioleoyl-rac-glycerol, 20.2 μg/ml phosphatidylserine, 100 μM syntide-2, and purified PKC from rat cerebral cortex. The reaction was started by adding PKC and activity determined by the phosphocellulose paper method. [1]
For autophosphorylation of CaM-kinase II, the kinase (1.1 μg/ml) was incubated at 30°C for 10 min in a reaction mixture containing 40 mM Hepes-NaOH (pH 8.0), 5 mM magnesium acetate, 0.2 mM CaCl2, 0.1 mM EGTA, 0.01% Tween 20, 1 μM calmodulin, and 50 μM [γ-32P]ATP with varying concentrations of inhibitor. The reaction was stopped by adding 22.7 mM EDTA, and 32P incorporation into the kinase was measured by the phosphocellulose paper method. [1]
In vitro enzyme assays for Autocamtide-2-Related Inhibitory Peptide are performed to measure its inhibition of CaMKII activity. A typical protocol involves incubating recombinant CaMKII with a peptide substrate (e.g., autocamtide-2) in the presence of ATP and Ca2+/calmodulin. The incorporation of radioactive phosphate into the substrate is measured, or a fluorescence-based assay is used. Various concentrations of the inhibitory peptide are added to the reaction, and the IC50 value of 40 nM is determined from the inhibition curve.

In vitro cellular assays for Autocamtide-2-Related Inhibitory Peptide are less common due to its limited cell permeability. However, it can be used in cell lysates or permeabilized cell systems to study CaMKII activity. In such assays, cells are treated with the peptide, and CaMKII activity is measured by immunoblotting for the phosphorylation of known CaMKII substrates. Alternatively, the peptide can be introduced into cells using methods such as microinjection or transfection to study its effects on cellular functions dependent on CaMKII.

Specific in vivo animal study protocols for Autocamtide-2-Related Inhibitory Peptide are not detailed in standard references, as it is primarily a tool for in vitro biochemistry. If used in vivo, it would require specialized delivery methods due to its peptide nature. Studies could involve infusing the peptide into specific brain regions using osmotic minipumps to study its effects on CaMKII-dependent behaviors or physiological processes. Such studies would confirm the role of CaMKII in various in vivo functions.

Specific pharmacokinetic data for Autocamtide-2-Related Inhibitory Peptide are not reported, as it is a research peptide and not a drug candidate. Its properties would be typical of a peptide, including poor oral bioavailability, rapid clearance, and a short half-life. Its use is largely confined to in vitro experiments where these limitations are not relevant. For any in vivo application, significant formulation or modification would be required to improve its stability and delivery.

Comprehensive toxicology data for Autocamtide-2-Related Inhibitory Peptide are not typically reported, as it is a research tool. Its toxicity would be related to its pharmacological effect of inhibiting CaMKII, a kinase involved in numerous essential cellular processes. Non-selective or excessive inhibition of CaMKII could lead to significant cellular dysfunction. However, the peptide's high selectivity minimizes off-target effects, which is a key feature for its use as a research tool. Standard safety assessments would be required for therapeutic development.

[1]. A novel highly specific and potent inhibitor of calmodulin-dependent protein kinase II. Biochem Biophys Res Commun. 1995 Jul 26;212(3):806-12.

AIP is a nonphosphorylatable analog of autocamtide-2, with Ala substituted for the phosphorylation site (Thr9). [1]
The inhibition by AIP is independent of Ca2+/calmodulin, and it binds to the substrate-binding site for the autophosphorylation site, which is distinct from the exogenous substrate (e.g., syntide-2) binding site. [1]
AIP does not inhibit the 30-kDa proteolytic fragment of CaM-kinase II in a calmodulin-competitive manner, unlike KN-93. [1]
AIP is suggested as a useful tool for studying the physiological roles of CaM-kinase II, being more potent and selective than previously available inhibitors such as KN-93 and CaMK-(281-302Ala286). [1]
Autocamtide-2-Related Inhibitory Peptide is a highly specific and potent inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), with an IC50 of 40 nM. It is active in both the presence and absence of Ca2+/calmodulin and is selective over other kinases such as PKC. The peptide is widely used in in vitro kinase assays to selectively block CaMKII activity, making it a valuable tool for studying calcium signaling pathways. It is not a drug but a standard research reagent for studying CaMKII function.

C65H114N20O21

1822.12084984779

1821.07

167114-91-2

Autocamtide-2-related inhibitory peptide TFA

Typically exists as solid at room temperature

8.469

[C@H](CCCNC(=N)N)(C(N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C)C(=O)N[C@H](C(=O)O)CC(C)C)=O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)NC(=O)CCCCCCCCCCCCC.C(F)(F)(F)C(=O)O

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)

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 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).

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)]
*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)

---

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
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

---

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.

---

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