Indolicidin acts primarily on bacterial cell membranes. Its mechanism involves high-affinity binding to lipopolysaccharide (LPS) on the outer membrane of Gram-negative bacteria, followed by permeabilization and disruption of the cytoplasmic membrane through the formation of voltage-dependent ion channels [1].

There are 13 amino acids in indolicidin, 5 of which are tryptophan residues, and formamidated arginine at the carboxyl-terminal. Of all known proteins, indolicidin has the highest tryptophan content. This special antibiotic peptide may function differently depending on the presence of several tryptophan residues. Tridecapeptide amide indolicidin has in vitro bactericidal activity similar to the strongest bacteriocin peptides or defensins [1]. The purified surface lipopolysaccharide binds to indolicidin with a high affinity, allowing it to penetrate E's outer membrane. coli to the small hydrophobic molecule 1-N-phenylnaphthylamine (Mr 200), which is in line with indolicidin's self-promoted uptake pathway crossing the outer membrane. When indoxidine's carboxyl terminus is methyl esterified, it becomes more effective against both Gram-positive and Gram-negative bacteria. This is linked to higher lipopolysaccharide binding and increased outer membrane permeability in Gram-negative bacteria. Indoxitin acts on the cytoplasmic membrane in Escherichia coli, and membrane permeabilization exposes cytoplasmic β-galactosidase for measurement [2].

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Indolicidin exhibited broad-spectrum antimicrobial activity in vitro. Minimum Inhibitory Concentration (MIC) values against various bacterial strains ranged from 4 μg/ml to 64 μg/ml. For example, MICs were 16 μg/ml for E. coli UB1005, 64 μg/ml for P. aeruginosa H103, 64 μg/ml for S. typhimurium 14028s, and 8 μg/ml for S. aureus RN4220. Its carboxyl-terminal methyl ester derivative (Indolicidin-C) showed increased potency, with MICs as low as 1-4 μg/ml against some strains [1].
Indolicidin bound to purified P. aeruginosa LPS with high affinity, comparable to polymyxin B, as determined by a dansyl-polymyxin B displacement assay. The concentration required to displace 50% of bound dansyl-polymyxin B (I₅₀) was used as a measure of relative affinity, though the exact I₅₀ value for indolicidin is not numerically stated in the text [1].
Indolicidin permeabilized the outer membrane of E. coli to the small hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN, M_r 200), with activity observed at concentrations as low as 3-5 μg/ml. However, it did not facilitate the uptake of the larger protein lysozyme (M_r 14,000), even at concentrations up to 70 μg/ml [1].
Indolicidin permeabilized the cytoplasmic (inner) membrane of E. coli ML-35, as assayed by the unmasking of cytoplasmic β-galactosidase activity using o-nitrophenyl-β-D-galactoside (ONPG) as a substrate. Permeabilization occurred rapidly (lag time <1 min) at concentrations as low as 4 μg/ml. This activity was significantly reduced in cells pretreated with the uncoupler carbonyl cyanide-m-chlorophenyl hydrazone (CCCP, 100 μM), indicating a dependence on the transmembrane electrical potential [1].
Indolicidin caused a voltage-dependent increase in transmembrane current in planar lipid bilayers (composed of phosphatidylcholine/phosphatidylserine, 5:1). A threshold potential of approximately -70 to -80 mV (trans-negative) was required to activate this current increase. The macroscopic current increase was attributed to the formation of discrete ion channels with single-channel conductances ranging from 0.05 to 0.15 nS [1].
Circular dichroism spectroscopy indicated that Indolicidin adopts a weak poly-L-proline II extended helix structure in aqueous solution, which becomes substantially more pronounced upon interaction with liposomes [1].

Outer Membrane Permeabilization (NPN Uptake Assay): E. coli UB1005 cells were grown to mid-log phase, harvested, washed, and resuspended in buffer (5 mM HEPES, pH 7.2, 5 mM KCN). The fluorescent probe NPN was added to the cell suspension at a final concentration of 10 μM. The baseline fluorescence (weak in aqueous medium) was measured. Upon addition of increasing concentrations of Indolicidin, the increase in fluorescence was monitored, which corresponds to NPN partitioning into the hydrophobic interior of the permeabilized outer membrane [1].
Cytoplasmic Membrane Permeabilization (β-Galactosidase Unmasking Assay): E. coli ML-35 (a strain constitutive for cytoplasmic β-galactosidase but lacking lactose permease) in logarithmic growth phase was harvested and resuspended in 10 mM sodium phosphate buffer (pH 7.4) containing 100 mM NaCl. The substrate ONPG was added. At time zero, different amounts of Indolicidin were added to the cell suspension. The hydrolysis of ONPG by the released cytoplasmic β-galactosidase was monitored spectrophotometrically at 420 nm by measuring the production of o-nitrophenol over time [1].

[1]. Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils. J Biol Chem. 1992 Mar 5;267(7):4292-5.

[2]. Mode of action of the antimicrobial peptide indolicidin. J Biol Chem. 1996 Aug 9;271(32):19298-303.

Indole-3-amino acid is a cationic antimicrobial peptide (ILPWKWPWWPWRR-NH2) composed of 13 amino acids, isolated from bovine neutrophils. It has a unique composition, containing 39% tryptophan and 23% proline, making it the smallest known linear natural antimicrobial peptide and the protein with the highest tryptophan content [1]. Its mechanism of action includes: 1) binding with high affinity to lipopolysaccharide (LPS) on the outer membrane of Gram-negative bacteria, promoting its own uptake; 2) crossing the outer membrane; 3) interacting with the cytoplasmic membrane, leading to the formation of voltage-dependent ion channels, thereby dissipating transmembrane potential and causing cell death [1]. Methyl esterification at the carboxyl terminus (generating indole-3-C) increases the net positive charge of the peptide, thereby enhancing its antimicrobial activity against most tested bacteria, increasing LPS binding affinity, and enhancing outer membrane permeability [1].
A certain membrane potential threshold is required for the formation of channels in a planar lipid bilayer. The channels formed vary in size, but once formed, they are relatively stable. Given its small molecular weight, the peptide may adopt an extended structure and aggregate to form these channels [1].

C100H132N26O13

1906.28448

1905.05

140896-21-5

Indolicidin acetate;Indolicidin TFA

16129733

White to off-white solid powder

11.317

23

17

50

139

4190

14

USSYUMHVHQSYNA-UHFFFAOYSA-N

InChI=1S/C100H132N26O13/c1-5-57(4)85(102)95(136)123-79(45-56(2)3)96(137)124-42-20-36-82(124)92(133)118-76(46-58-51-110-68-28-11-6-23-63(58)68)89(130)116-74(33-16-17-39-101)88(129)121-80(49-61-54-113-71-31-14-9-26-66(61)71)97(138)125-43-21-38-84(125)94(135)120-78(48-60-53-112-70-30-13-8-25-65(60)70)91(132)122-81(50-62-55-114-72-32-15-10-27-67(62)72)98(139)126-44-22-37-83(126)93(134)119-77(47-59-52-111-69-29-12-7-24-64(59)69)90(131)117-75(35-19-41-109-100(106)107)87(128)115-73(86(103)127)34-18-40-108-99(104)105/h6-15,23-32,51-57,73-85,110-114H,5,16-22,33-50,101-102H2,1-4H3,(H2,103,127)(H,115,128)(H,116,130)(H,117,131)(H,118,133)(H,119,134)(H,120,135)(H,121,129)(H,122,132)(H,123,136)(H4,104,105,108)(H4,106,107,109)

1-[2-[[6-amino-2-[[2-[[1-[2-[(2-amino-3-methylpentanoyl)amino]-4-methylpentanoyl]pyrrolidine-2-carbonyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]hexanoyl]amino]-3-(1H-indol-3-yl)propanoyl]-N-[1-[[1-[2-[[1-[[1-[(1-amino-5-carbamimidamido-1-oxopentan-2-yl)amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]carbamoyl]pyrrolidin-1-yl]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]pyrrolidine-2-carboxamide

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)

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.)