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| Targets |
IC50: skin-antimicrobial peptide (SAP)[1]
The primary target of human beta-defensin-1 is the cell membrane of a wide range of bacteria. The cationic peptide interacts electrostatically with negatively charged components of the bacterial cell envelope, such as lipopolysaccharide (LPS) in Gram-negative bacteria or lipoteichoic acid (LTA) in Gram-positive bacteria. This interaction leads to the disruption of the bacterial membrane, causing pore formation, loss of membrane potential, leakage of cellular contents, and ultimately bacterial cell death. This mechanism of action is non-specific and rapid, making it difficult for bacteria to develop resistance. HbetaD-1 also has immunomodulatory functions, interacting with various immune receptors, including chemokine receptors, to shape the adaptive immune response. |
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| ln Vitro |
In vitro, human beta-defensin-1 demonstrates potent antimicrobial activity against a broad spectrum of bacteria, including both Gram-positive and Gram-negative species. Its activity is often measured as a minimum inhibitory concentration (MIC), which can range from 2-50 ug/mL depending on the specific bacterial strain. HbetaD-1 has been shown to be particularly effective against sperm-associated bacteria, which are a common cause of infertility. Its antimicrobial activity is salt-sensitive, as high salt concentrations can reduce its positive charge and thus its ability to bind bacterial membranes. The peptide also shows some activity against fungi, such as Candida albicans, and has been reported to have antiviral activity against certain enveloped viruses.
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| ln Vivo |
The in vivo activity of human beta-defensin-1 is difficult to assess as it is not a drug candidate for systemic administration. Its role has been studied using HbetaD-1 knockout mice, which are more susceptible to bacterial infections. In mouse models of skin infection, topical application or local injection of recombinant HbetaD-1 can reduce bacterial loads. In models of inflammatory bowel disease, HbetaD-1 expression is often altered, suggesting it plays a role in modulating the gut microbiome and mucosal immunity. However, detailed in vivo studies are limited. The expression of HbetaD-1 itself is known to be regulated by various factors, including commensal bacteria, inflammatory cytokines (e.g., TNF-alpha, IL-1beta), and bacterial products like LPS, which are often used in in vivo models to induce its expression.
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| Enzyme Assay |
A typical non-cellular antimicrobial assay for HbetaD-1 is the broth microdilution method. A standard inoculum of bacteria (e.g., E. coli ATCC 25922, 5 × 10⁵ CFU/mL) is prepared in cation-adjusted Mueller-Hinton broth (MHB). Serial two-fold dilutions of HbetaD-1 (e.g., 0.5-128 ug/mL) are made in MHB. An equal volume (100 uL) of bacterial suspension and peptide solution is mixed in a 96-well plate and incubated at 37degC for 18-24 hours. The MIC is the lowest concentration of peptide that inhibits visible bacterial growth, as determined by absorbance at 600 nm. A standard curve is used to calculate the concentration of bacteria.
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| Cell Assay |
In vitro cell-based assays for HbetaD-1 are less common due to its membrane-disrupting activity, but the peptide's cytotoxicity can be assessed using human cells. Human skin fibroblasts (HSF) or keratinocytes (e.g., HaCaT cells) are cultured in DMEM with 10% FBS. Cells are seeded in 96-well plates at 1 × 10⁴ cells/well and allowed to adhere overnight. The medium is then replaced with fresh medium containing HbetaD-1 at various concentrations (0-100 ug/mL). After 24-48 hours of incubation, cell viability is measured by the MTT assay. The CC₅0 (50% cytotoxic concentration) is calculated. This assay helps determine the therapeutic index of the peptide by comparing the MIC values against bacteria to the CC₅0 in human cells.
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| Animal Protocol |
There is no standard protocol for in vivo animal studies for HbetaD-1 as a therapeutic, but it can be studied in a mouse model of skin infection. 6-8 week old female BALB/c mice are anesthetized, and their dorsal skin is shaved and depilated. A superficial wound is made using a sterile needle or by tape stripping. An inoculum of 1 × 10⁷ CFU of S. aureus or E. coli is applied topically. After 2 hours, the wound is treated with a topical formulation (e.g., a cream or gel) containing HbetaD-1 (0.1-1 mg/mL) once daily for 3 days. A vehicle control group is included. At the end of the study, mice are euthanized, and a punch biopsy of the infected skin is taken. The skin sample is homogenized, and serial dilutions are plated to enumerate CFU per gram of tissue. The bacterial load is compared between the treatment and vehicle groups.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties of HbetaD-1 are characteristic of a peptide. It is a peptide of 36 amino acids (MW ~ 4000 Da). HbetaD-1 is not suitable for oral administration due to degradation in the gastrointestinal tract. If administered intravenously, it has a very short plasma half-life (t1/2), typically on the order of minutes, due to rapid proteolytic degradation and renal clearance. Its large size and hydrophilicity prevent it from crossing the blood-brain barrier. Its endogenous expression is tightly regulated by the host, and it is not considered to have systemic bioavailability when administered topically. Therefore, it is not developed as a systemic therapeutic.
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| Toxicity/Toxicokinetics |
No acute or chronic toxicity data is available for HbetaD-1 as a therapeutic agent. As an endogenous peptide, it is considered safe by the body, but high concentrations could be toxic. In vitro cytotoxicity data suggests it has a low CC₅0 in mammalian cells, indicating a window for topical use. Since it is part of the innate immune system, it is not immunogenic in its native form. However, if produced recombinantly, it could have impurities that cause reactions. Standard safety precautions for handling peptides should be followed. Its toxicological profile is a subject of ongoing research for potential therapeutic applications.
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| References | |
| Additional Infomation |
Human beta-defensin-1 is not an approved drug. It is a naturally occurring human antimicrobial peptide with potential therapeutic applications for infectious diseases, immune modulation, and fertility. Its mechanism of action is primarily through the disruption of bacterial cell membranes, making it a promising candidate for an antibiotic that may circumvent resistance. While not an approved drug, it is a standard research chemical used to study the innate immune system and has been investigated in clinical contexts related to skin diseases (e.g., atopic dermatitis) and inflammatory bowel disease. No clinical trials have been registered for its use as a drug, though it serves as a reference standard. For research use only; not for human therapeutic or diagnostic use.
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| Molecular Formula |
C167H256N48O50S6
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|---|---|
| Molecular Weight |
3928.53
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| Exact Mass |
3926.732
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| CAS # |
452274-53-2
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| PubChem CID |
171921199
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| Appearance |
White to off-white solid powder
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| Hydrogen Bond Donor Count |
56
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| Hydrogen Bond Acceptor Count |
63
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| Rotatable Bond Count |
64
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| Heavy Atom Count |
271
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| Complexity |
9120
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| Defined Atom Stereocenter Count |
36
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| SMILES |
CC[C@H](C)[C@H]1C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)NCC(=O)N[C@H](C(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CSSC[C@@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)NCC(=O)NCC(=O)N[C@H](C(=O)N3)CCC(=O)N)CO)CO)C(C)C)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC4=CC=C(C=C4)O)NC(=O)[C@H](CC5=CNC=N5)NC(=O)[C@H](CC(=O)O)N)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC2=O)CC6=CC=C(C=C6)O)CCCNC(=N)N)CCCCN)C)CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)C(=O)N7CCC[C@H]7C(=O)N1)C)CO)CC8=CC=C(C=C8)O)CC(C)C)[C@@H](C)O)CCC(=O)N)[C@@H](C)CC)CCCCN)[C@@H](C)O)CC9=CC=CC=C9
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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 | 0.2545 mL | 1.2727 mL | 2.5455 mL | |
| 5 mM | 0.0509 mL | 0.2545 mL | 0.5091 mL | |
| 10 mM | 0.0255 mL | 0.1273 mL | 0.2545 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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