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| 5mg |
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| 10mg |
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| 25mg |
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| 50mg |
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| 100mg |
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| 250mg |
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| Other Sizes |
Purity: ≥98%
| Targets |
TPT-260 targets the retromer core complex, specifically binding at the interface between Vps35 and Vps29 proteins (site 2). [1]
Binding affinity: Kd for the retromer core complex is approximately 5 μM as determined by microscale thermophoresis. [1] Thermal stabilization: increases the melting temperature (Tm) of the reconstituted Vps26-Vps35-Vps29 heterotrimer from 49 °C to 59.5 °C (ΔTm = +10.5 °C) at the highest concentration tested. [1] |
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| ln Vitro |
In vitro, TPT-260 (R55) stabilizes the purified reconstituted retromer core complex (Vps26-Vps35-Vps29) against thermal denaturation as measured by differential scanning fluorimetry (DSF), increasing the melting temperature by approximately 10 °C. The compound has no effect on the thermal unfolding of individual Vps35 or Vps29 proteins, indicating specific binding at the interface. The Q538W mutation on Vps35 blocks the binding site and abolishes the stabilizing effect. [1]
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| ln Vivo |
TPT-260 (5 μM, 48 h) increases retromer protein levels in primary cortical neurons: Vps35 by ~80% (p < 0.001) and Vps26 by ~50% (p < 0.001) vs vehicle, with no change in mRNA levels. [1]
In hippocampal neurons, TPT-260 reduces endogenous mouse Aβ40 (~24%, p < 0.001) and Aβ42 (~28%, p < 0.001). In J20 transgenic mouse neurons (human APP mutant), it reduces human Aβ40 (~44%, p < 0.01) and Aβ42 (~39%, p < 0.01). IC50 for Aβ reduction ~12 μM. [1] TPT-260 reduces BACE1-dependent APP fragments: β-CTF by ~70% (p < 0.001) and sAPPβ by ~25% (p < 0.001), and increases α-secretase fragment sAPPα by ~90% (p < 0.05). [1] In hippocampal neurons, TPT-260 decreases colocalization of APP with early endosomal marker EEA1 (p < 0.05) and of SorL1 with EEA1 (p < 0.01), indicating enhanced retromer-mediated trafficking out of endosomes. [1] In Vps26 knockdown neurons (Vps26flox/flox with Cre lentivirus, ~50% knockdown), TPT-260 fails to reduce Aβ42 levels, confirming that the Aβ-lowering effect is retromer-dependent. [1] |
| Enzyme Assay |
The thermal stabilization assay (differential scanning fluorimetry, DSF) was performed using purified recombinant retromer complex (Vps26-Vps35-Vps29) at 2 μM in buffer containing 50 mM NaH₂PO₄, 300 mM NaCl, pH 8.0, with 20× Sypro orange dye. Compounds were added at varying concentrations. Melting curves were determined by reading fluorescence in 0.3 °C intervals from 25 °C to 95 °C. The melting temperature (Tm) was taken as the inflection point of the melting curve. [1]
Microscale thermophoresis was used to determine the binding affinity (Kd) of TPT-260 for the retromer core complex. The Kd was found to be approximately 5 μM with a negative thermophoretic trace. [1] In silico docking: The Vps29-Vps35 crystal structure (PDB 2R17) was submitted to Q-site Finder to identify binding pockets. Virtual screening was performed using Glide (Schrödinger) against a library of 46,000 lead-like compounds. Ten docking sites were identified; six at the interface. Compound R55 was predicted to bind at site 2, the largest pocket at the Vps35-Vps29 interface. [1] |
| Cell Assay |
Primary hippocampal and cortical neurons were cultured from wild-type mice or J20 transgenic mice (carrying human APP with Swedish and Indiana mutations). Cells were grown for 14 days in vitro. TPT-260 (or vehicle, 50% DMSO in water) was applied at 11 DIV at indicated concentrations (typically 5 μM) for 48 h before analysis. Cell viability was assessed using XTT colorimetric assay; doses from 0.5 μM to 50 μM were nontoxic. [1]
For immunocytochemistry, primary hippocampal neurons on coverslips were fixed in 2% PFA/0.5% sucrose, permeabilized with 0.1% Triton X-100 (for APP) or 0.05% saponin (for Vps35 and SorL1), and stained with anti-APP (C-terminal), anti-EEA1, anti-MAP2, or anti-SorL1 antibodies. Confocal images were captured with a Zeiss LSM 700 META microscope, and colocalization was analyzed using ImageJ plugins (Colocalization Finder or JACoB). [1] For Vps26 knockdown experiments, primary neurons from Vps26flox/flox mice were infected at DIV5 with lentivirus expressing active Cre recombinase (CRE) or catalytically dead Cre (dead-Cre) for 10 days. Knockdown efficiency (~50%) was confirmed by western blot. [1] |
| ADME/Pharmacokinetics |
Cell viability studies using a colorimetric XTT assay in primary cortical neurons showed that TPT-260 at concentrations ranging from 0.5 μM to 50 μM was nontoxic to neurons over 48 h (data not shown). [1]
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| Toxicity/Toxicokinetics |
TPT-260 (R55) is a thiophene thiourea derivative. It acts as a pharmacological chaperone by binding to the Vps35-Vps29 interface of the retromer core complex, thereby stabilizing the entire trimeric complex and protecting it from degradation. This enhances retromer-mediated trafficking of APP and SorL1 out of endosomes, reducing BACE1-dependent processing of APP. The compound increases retromer protein levels and shifts APP processing toward the neuroprotective α-secretase pathway, generating a fragment profile similar to that of a protective APP mutation (A673T). The effect is retromer-dependent as shown by Vps26 knockdown experiments. This study demonstrates for the first time that small molecules can act as pharmacological chaperones for a multiprotein complex. TPT-260 may have therapeutic potential for Alzheimer's disease and other retromer-related neurodegenerative diseases (e.g., Parkinson's disease). [1]
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| References |
Nat Chem Biol.2014Jun;10(6):443-9.
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| Additional Infomation |
[5-(Carbamimidoylsulfanylmethyl)thiophen-2-yl]methyl carbamimidothioate is an organic molecular entity.
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| Molecular Formula |
C8H12N4S3
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|---|---|
| Molecular Weight |
260.4027
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| Exact Mass |
260.022
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| CAS # |
769856-81-7
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| Related CAS # |
TPT-260 Dihydrochloride;2076-91-7
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| PubChem CID |
414147
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.6±0.1 g/cm3
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| Boiling Point |
436.9±55.0 °C at 760 mmHg
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| Flash Point |
218.0±31.5 °C
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| Vapour Pressure |
0.0±1.0 mmHg at 25°C
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| Index of Refraction |
1.770
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| LogP |
0.48
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
15
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| Complexity |
222
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| Defined Atom Stereocenter Count |
0
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| SMILES |
S1C(C([H])([H])S/C(=N/[H])/N([H])[H])=C([H])C([H])=C1C([H])([H])S/C(=N/[H])/N([H])[H]
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| InChi Key |
DSODRWWHAUGSGD-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C8H12N4S3.2ClH/c9-7(10)13-3-5-1-2-6(15-5)4-14-8(11)12;;/h1-2H,3-4H2,(H3,9,10)(H3,11,12);2*1H
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| Chemical Name |
thiophene-2,5-diylbis(methylene) dicarbamimidothioate dihydrochloride
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| Synonyms |
NSC-55712; NSC55712; NSC 55712; TPT260; TPT-260; TPT 260; TPT-260 HCl; TPT-260 2HCl; TPT-260 dihydrchloride; R55.
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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 |
| 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 | 3.8402 mL | 19.2012 mL | 38.4025 mL | |
| 5 mM | 0.7680 mL | 3.8402 mL | 7.6805 mL | |
| 10 mM | 0.3840 mL | 1.9201 mL | 3.8402 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.
 Identification of a retromer stabilizing pharmacological chaperone.Nat Chem Biol.2014Jun;10(6):443-9. |
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 The pharmacological chaperone increases retromer levels in neurons.Nat Chem Biol.2014Jun;10(6):443-9. |
 The pharmacological chaperone decreases Aβ peptide accumulation and reduces the pathogenic pathway of APP.Nat Chem Biol.2014Jun;10(6):443-9. |
 The pharmacological chaperone shifts the endosomal localization of APP and SorL1.Nat Chem Biol.2014Jun;10(6):443-9. |
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 The pharmacological chaperone specifically modulates Aβ production through the retromer pathway.
Characterization of the weak link in retromer complex stability.Nat Chem Biol.2014Jun;10(6):443-9. |
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