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| Targets |
The primary target of STh is guanylate cyclase C (GC-C), a transmembrane receptor expressed on the apical surface of intestinal epithelial cells. Upon binding to GC-C, STh activates the receptor's intrinsic guanylate cyclase activity, converting GTP to cGMP. Elevated cGMP levels activate protein kinase G (PKG) and downstream effectors, leading to phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel, increased chloride secretion, and inhibition of sodium absorption. This electrolyte imbalance results in secretory diarrhea. STh serves as a potent and selective GC-C agonist with high affinity (KD in the low nanomolar range).
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| ln Vitro |
In vitro studies demonstrate that STh (1-100 nM) stimulates cGMP production in T84 human colonic epithelial cells and other GC-C-expressing cell lines in a dose-dependent manner. The peptide exhibits stability in the presence of heat (100degC for 30 minutes) and resistance to proteolytic degradation due to its compact disulfide-bridged structure. STh has been used in Ussing chamber studies to measure short-circuit current (Isc) as an indicator of chloride secretion across intestinal epithelial monolayers, with EC50 values typically in the 1-10 nM range. The peptide also activates cGMP-dependent signaling pathways (PKG, VASP phosphorylation) and inhibits proliferation in GC-C-expressing cancer cell lines, suggesting potential roles beyond diarrhea pathogenesis.
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| ln Vivo |
In vivo, STh induces fluid accumulation in the mouse intestinal loop model, a standard assay for enterotoxin activity. Administration of STh (1-10 microg per loop) into ligated intestinal segments of anesthetized mice leads to significant fluid secretion within 4-6 hours, measured as the ratio of fluid volume to loop length (mL/cm). In suckling mouse assays, oral administration of STh (0.1-1 microg) results in diarrhea and fluid accumulation in the intestines. The toxin has been studied in piglet models of ETEC infection, where it contributes to the characteristic secretory diarrhea observed in infected animals. STh also serves as a tool for vaccine development, as immunization with non-toxic STh derivatives or toxoids can induce protective antibodies against ETEC challenge in animal models.
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| Enzyme Assay |
Non-cell-based binding assays can be performed using competitive ELISA or surface plasmon resonance (SPR) to measure STh binding to GC-C receptor. For ELISA, microtiter plates are coated with recombinant GC-C extracellular domain (ECD) protein. Serially diluted STh (1 pM to 1 microM) is added, and bound peptide is detected using anti-STh primary antibody and HRP-conjugated secondary antibody. For SPR, biotinylated STh is immobilized on a streptavidin sensor chip, and GC-C-ECD protein is flowed over the surface at increasing concentrations (0.1-100 nM) to determine binding kinetics (ka, kd, KD). Alternatively, radio-ligand binding assays using 125I-labeled STh (specific activity 2000 Ci/mmol) can quantify receptor binding affinity with KD typically reported as 0.5-2 nM.
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| Cell Assay |
Cells expressing GC-C (e.g., T84, Caco-2, or GC-C-transfected HEK293 cells) are cultured in DMEM/F12 or RPMI medium supplemented with 10% FBS at 37degC in 5% CO2. Cells are seeded in 24-well plates (2×10⁵ cells/well) and allowed to grow for 48 hours. For cGMP measurement, cells are incubated with STh (0.01-1000 nM) in the presence of 0.5 mM IBMX (phosphodiesterase inhibitor) for 30 minutes at 37degC. The reaction is stopped by removing medium and adding 0.1 M HCl. Cell lysates are collected and centrifuged, and supernatants are assayed for cGMP using a competitive ELISA kit. For chloride secretion assays, T84 cells are grown on permeable filter supports (Transwell inserts, 0.4 microm pore size) for 14-21 days until confluent monolayers with high transepithelial electrical resistance (TEER >1000 omega·cm2) are established. Monolayers are mounted in Ussing chambers, and STh (1-100 nM) is added to the apical side. Short-circuit current (Isc) is measured as an indicator of electrogenic chloride secretion.
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| Animal Protocol |
Mouse ligated ileal loop model: Female BALB/c mice (6-8 weeks old, 18-22 g) are anesthetized with ketamine/xylazine. A midline laparotomy is performed, and a 3-4 cm segment of ileum is ligated with silk suture, taking care to preserve the mesenteric blood supply. STh is dissolved in PBS (1-10 microg per loop in 100-200 microL volume) and injected into the ligated loop. Negative control loops receive PBS alone. The abdomen is closed with sutures, and mice are allowed to recover for 4-6 hours. Mice are then euthanized, and the intestinal loops are excised. Loop length (cm) and fluid volume (mL) are measured, and the fluid accumulation ratio (mL/cm) is calculated. For suckling mouse assay: 2-4 day old suckling mice are fasted for 2 hours, then orally gavaged with STh (0.1-1 microg in 50 microL of 2% Evans blue dye solution). After 3 hours at room temperature, mice are euthanized, and the entire intestine is removed. The intestine-to-carcass weight ratio is calculated as an index of fluid accumulation. For vaccine studies, mice are immunized subcutaneously or intranasally with STh toxoids or conjugated derivatives (10-50 microg per dose) with adjuvant (e.g., cholera toxin or aluminum hydroxide), followed by booster doses at 2-4 week intervals. Serum and fecal antibody titers are measured by ELISA, and protection against challenge with live ETEC bacteria or purified STh is assessed.
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| ADME/Pharmacokinetics |
As a peptide, STh has a short plasma half-life (minutes) when administered intravenously due to rapid proteolytic degradation and renal clearance. The three disulfide bridges provide stability against heat and some proteases but not against serum proteases. For vaccine applications, STh is often conjugated to carrier proteins (e.g., bovine serum albumin, keyhole limpet hemocyanin) or formulated with adjuvants to enhance immunogenicity and extend exposure. In animal models, STh doses used for toxin challenge are typically 1-10 microg per animal. Oral delivery of STh is feasible due to its stability in the gastrointestinal tract, but systemic absorption is minimal. Pharmacokinetic parameters such as volume of distribution, clearance, and half-life are not well-defined due to its primary use as a toxin in acute challenge models rather than as a therapeutic agent.
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| Toxicity/Toxicokinetics |
STh is an enterotoxin and is considered a virulence factor, causing secretory diarrhea in infected hosts. In animal models, administration of native STh at doses ≥1 microg induces significant fluid secretion and diarrhea without systemic toxicity. The LD50 of STh in suckling mice is estimated >100 microg, indicating a wide safety margin between diarrheagenic and lethal doses. STh is not cytotoxic to intestinal epithelial cells at concentrations ≤100 nM; rather, it activates physiologic signaling pathways without causing cell death. No genotoxicity, carcinogenicity, or reproductive toxicity data are available for STh. As a vaccine antigen, detoxified STh derivatives (e.g., heat-inactivated or genetically detoxified mutants) are used to avoid toxic effects, and they are well-tolerated in animal immunization studies, with no adverse effects reported.
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| References |
[1]. Pål PUNTERVOLL, et al. Heat-stable enterotoxins mutants as antidiarrheal vaccine antigens. WO2020039387A1.
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| Additional Infomation |
STh is available as a research reagent for studying ETEC pathogenesis, intestinal physiology, and GC-C signaling. The peptide sequence is NSSNYCCELCCNPACTGCY, with three disulfide bridges (Cys6-11, Cys7-15, Cys10-18) essential for biological activity. STh is also known as STa (heat-stable enterotoxin a) to distinguish it from STb (a different heat-stable toxin). The peptide is a key component of ETEC vaccines under development, including several candidates in phase I-III clinical trials. However, STh itself is not an FDA-approved vaccine or therapeutic; it serves as a model antigen for evaluating vaccine platforms. The compound is stored as a lyophilized powder at -20degC and is soluble in water or PBS. Care should be taken when handling STh due to its enterotoxic activity; use of gloves and proper disposal of contaminated materials is recommended.
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| Molecular Formula |
C79H112N22O30S6
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|---|---|
| Molecular Weight |
2042.25
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| Exact Mass |
2040.623
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| CAS # |
118447-40-8
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| PubChem CID |
168013072
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
-11.9
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| Hydrogen Bond Donor Count |
28
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| Hydrogen Bond Acceptor Count |
37
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| Rotatable Bond Count |
31
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| Heavy Atom Count |
137
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| Complexity |
4350
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| Defined Atom Stereocenter Count |
19
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| SMILES |
C[C@H]1C(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CSSC[C@H](NC(=O)CNC(=O)[C@@H](NC2=O)[C@@H](C)O)C(=O)N[C@@H](CC4=CC=C(C=C4)O)C(=O)O)C(=O)N[C@@H](CSSC[C@@H](C(=O)N3)NC(=O)[C@H](CC5=CC=C(C=C5)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)N)N)C(=O)N[C@H](C(=O)N6CCC[C@H]6C(=O)N1)CC(=O)N)CC(C)C)CCC(=O)O
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| InChi Key |
ALQPDZQRJKSEQZ-MZBYGZHOSA-N
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| InChi Code |
InChI=1S/C79H112N22O30S6/c1-33(2)18-42-65(116)96-52-30-133-132-27-49(70(121)92-46(79(130)131)20-37-9-13-39(106)14-10-37)86-59(110)24-84-77(128)61(35(4)104)100-75(126)54-32-137-134-28-50(71(122)87-41(64(115)88-42)15-16-60(111)112)98-74(125)53(31-136-135-29-51(99-73(52)124)72(123)91-45(23-58(83)109)78(129)101-17-5-6-55(101)76(127)85-34(3)62(113)95-54)97-66(117)43(19-36-7-11-38(105)12-8-36)89-67(118)44(22-57(82)108)90-68(119)48(26-103)94-69(120)47(25-102)93-63(114)40(80)21-56(81)107/h7-14,33-35,40-55,61,102-106H,5-6,15-32,80H2,1-4H3,(H2,81,107)(H2,82,108)(H2,83,109)(H,84,128)(H,85,127)(H,86,110)(H,87,122)(H,88,115)(H,89,118)(H,90,119)(H,91,123)(H,92,121)(H,93,114)(H,94,120)(H,95,113)(H,96,116)(H,97,117)(H,98,125)(H,99,124)(H,100,126)(H,111,112)(H,130,131)/t34-,35+,40-,41-,42-,43-,44-,45-,46-,47-,48-,49-,50-,51-,52-,53-,54-,55-,61-/m0/s1
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| Chemical Name |
(2S)-2-[[(1R,4S,7S,13S,16R,21R,24R,27S,30S,33R,38R,44S)-21-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2,4-diamino-4-oxobutanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-4-oxobutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-13-(2-amino-2-oxoethyl)-27-(2-carboxyethyl)-44-[(1R)-1-hydroxyethyl]-4-methyl-30-(2-methylpropyl)-3,6,12,15,22,25,28,31,40,43,46,51-dodecaoxo-18,19,35,36,48,49-hexathia-2,5,11,14,23,26,29,32,39,42,45,52-dodecazatetracyclo[22.22.4.216,33.07,11]dopentacontane-38-carbonyl]amino]-3-(4-hydroxyphenyl)propanoic 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, 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 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 | 0.4897 mL | 2.4483 mL | 4.8966 mL | |
| 5 mM | 0.0979 mL | 0.4897 mL | 0.9793 mL | |
| 10 mM | 0.0490 mL | 0.2448 mL | 0.4897 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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