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| Targets |
ComD2 (histidine kinase receptor of the ComD family). CSP-2 is a quorum-sensing signal peptide that binds to and activates its cognate receptor, ComD2, a membrane-bound histidine kinase. Activation of ComD2 leads to autophosphorylation and subsequent phosphotransfer to the response regulator ComE, which regulates transcription of competence genes (e.g., comX, comW, comCDE operon) and virulence factors. This signaling cascade induces competence development for natural transformation, biofilm formation, and virulence gene expression. CSP-2 is a 17-amino acid peptide: EMRISRIILDFLFLRKK.
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| ln Vitro |
CSP-2 is a quorum-sensing signal peptide that activates the ComD2 receptor. In vitro, CSP-2 at nanomolar concentrations induces competence development in S. pneumoniae, as measured by expression of competence genes (e.g., comX) and by transformation efficiency. It has an EC50 of 50.7 nM for its compatible receptor ComD2. The peptide also regulates biofilm formation, adherence to host cells, and production of virulence factors such as pneumolysin.
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| ln Vivo |
In vivo, CSP-2 is a key signaling molecule that enables S. pneumoniae to coordinate population density-dependent behaviors, including colonization, biofilm formation, and virulence. It is involved in the transition from colonization to invasive disease. In animal models, the competence system contributes to nasal colonization and lung infection. CSP-2 can be used to study quorum-sensing disruption as an anti-virulence strategy.
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| Enzyme Assay |
CSP-2 activates ComD2. For cell-free receptor binding assays, the membrane fraction of S. pneumoniae or HEK293 cells expressing ComD2 is prepared. Radiolabeled CSP-2 (e.g., 125I-labeled or 3H-labeled) at 0.1-10 nM is incubated with membrane preparations (50-200 microg protein) in binding buffer (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mg/mL BSA) at 25degC for 60 minutes. Non-specific binding is determined in the presence of 10 microM unlabeled CSP-2. Bound radioactivity is separated by rapid filtration through GF/B filters and counted by liquid scintillation or gamma counter. The dissociation constant (Kd) and Bmax are calculated from saturation binding curves. The EC50 for activation (50.7 nM) is typically determined in cell-based assays rather than cell-free binding assays.
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| Cell Assay |
For functional activation assays, S. pneumoniae strains containing a lux reporter under control of the comX promoter are used. Bacteria are cultured in competence medium (CAT medium) at 37degC to mid-log phase (OD600 ~0.2-0.3). CSP-2 is serially diluted (0.1 nM-10 uM) and added to the bacterial culture. After 30-60 minutes of incubation at 37degC, luminescence is measured using a plate reader. The EC50 for ComD2 activation is calculated by nonlinear regression (EC50 = 50.7 nM). Alternatively, transformation efficiency is measured: S. pneumoniae is treated with CSP-2 (10-100 ng/mL) for 15-20 minutes, followed by addition of 1 ug/mL of donor DNA (e.g., streptomycin resistance marker). After incubation at 37degC for 30-60 minutes, cells are plated on selective and non-selective media. Competence is expressed as the ratio of transformants to total viable cells. For biofilm formation, S. pneumoniae is grown in polystyrene 96-well plates in the presence or absence of CSP-2 (10-100 nM). After 24 hours of anaerobic incubation, biofilms are stained with crystal violet and quantified by measuring absorbance at 590 nm.
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| Animal Protocol |
In a murine nasopharyngeal colonization model, 6-8 week old female C57BL/6 mice are inoculated intranasally with 10⁷-10⁸ CFU of S. pneumoniae (e.g., D39 or TIGR4 strain) in 20-30 uL of PBS. In some experiments, CSP-2 (10-100 ug) is co-administered or administered separately to evaluate its effect on colonization. At various time points post-inoculation (1, 3, 5, 7 days), mice are euthanized, and nasopharyngeal washes are collected. Bacterial burden is determined by serial dilution and plating on blood agar. To study competence induction in vivo, mice are infected with S. pneumoniae, and nasopharyngeal samples are analyzed for expression of competence genes by RT-qPCR. CSP-2 is used as a tool to activate competence and study its role in pneumococcal pathogenesis.
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| ADME/Pharmacokinetics |
No pharmacokinetic data has been reported. As a 17-amino acid peptide (molecular weight 2178.69 g/mol), CSP-2 is a signaling molecule produced endogenously by S. pneumoniae in response to population density. Exogenously administered CSP-2 is a peptide that is likely degraded by proteases in vivo and has a very short half-life (minutes). It is not administered as a systemic drug but as a research tool in bacterial culture or localized infection models.
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| Toxicity/Toxicokinetics |
No specific toxicity data has been reported. CSP-2 is a peptide signaling molecule produced by S. pneumoniae and is not toxic to mammalian cells at the concentrations used for bacterial studies. It is not intended for administration to animals or humans outside of research contexts. Standard peptide handling precautions should be used.
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| References |
[1]. Yifang Yang, et al. Structural Characterization of Competence-Stimulating Peptide Analogues Reveals Key Features for ComD1 and ComD2 Receptor Binding in Streptococcus pneumonia. Biochemistry. 2018 Sep 11;57(36):5359-5369.
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| Additional Infomation |
CSP-2 is a 17-amino acid peptide with the sequence (single-letter): EMRISRIILDFLFLRKK. The molecular formula is C101H172N28O23S, and the molecular weight is 2178.69. It is a quorum-sensing signal peptide produced by S. pneumoniae (pneumococcus). It is used as a research tool to study bacterial quorum sensing, competence development, and virulence regulation. It is not approved for clinical use. It should be stored as a powder at -80degC under nitrogen. Purity: ≥98%.
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| Molecular Formula |
C101H172N28O23S
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| Molecular Weight |
2178.6852016449
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| Exact Mass |
2178.29
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| CAS # |
1174553-84-4
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| PubChem CID |
155514280
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
-5.2
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| Hydrogen Bond Donor Count |
32
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| Hydrogen Bond Acceptor Count |
30
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| Rotatable Bond Count |
81
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| Heavy Atom Count |
153
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| Complexity |
4420
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| Defined Atom Stereocenter Count |
20
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| SMILES |
CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(=O)O)N
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| InChi Key |
AQHPEKJLEFLYAT-IPQDWWCBSA-N
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| InChi Code |
InChI=1S/C101H172N28O23S/c1-14-58(10)79(127-86(139)66(37-28-45-112-100(107)108)116-85(138)68(41-47-153-13)114-82(135)63(104)39-40-77(131)132)96(149)126-76(54-130)94(147)118-67(38-29-46-113-101(109)110)87(140)128-81(60(12)16-3)97(150)129-80(59(11)15-2)95(148)125-72(50-57(8)9)90(143)124-75(53-78(133)134)93(146)123-74(52-62-32-21-18-22-33-62)92(145)121-71(49-56(6)7)89(142)122-73(51-61-30-19-17-20-31-61)91(144)120-70(48-55(4)5)88(141)117-65(36-27-44-111-99(105)106)83(136)115-64(34-23-25-42-102)84(137)119-69(98(151)152)35-24-26-43-103/h17-22,30-33,55-60,63-76,79-81,130H,14-16,23-29,34-54,102-104H2,1-13H3,(H,114,135)(H,115,136)(H,116,138)(H,117,141)(H,118,147)(H,119,137)(H,120,144)(H,121,145)(H,122,142)(H,123,146)(H,124,143)(H,125,148)(H,126,149)(H,127,139)(H,128,140)(H,129,150)(H,131,132)(H,133,134)(H,151,152)(H4,105,106,111)(H4,107,108,112)(H4,109,110,113)/t58-,59-,60-,63-,64-,65-,66-,67-,68-,69-,70-,71-,72-,73-,74-,75-,76-,79-,80-,81-/m0/s1
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| Chemical Name |
(2S)-6-amino-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-4-carboxybutanoyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylpentanoyl]amino]-3-hydroxypropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylpentanoyl]amino]-3-methylpentanoyl]amino]-4-methylpentanoyl]amino]-3-carboxypropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]-5-carbamimidamidopentanoyl]amino]hexanoyl]amino]hexanoic acid
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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. |
| 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 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.) |
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.4590 mL | 2.2950 mL | 4.5899 mL | |
| 5 mM | 0.0918 mL | 0.4590 mL | 0.9180 mL | |
| 10 mM | 0.0459 mL | 0.2295 mL | 0.4590 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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