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| Targets |
Retro 2 targets microtubule-dependent retrograde transport (intracellular trafficking pathway), [1][2][3]
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| ln Vitro |
In HeLa cells expressing GFP-LC3, retro-2 (1 μM; 1-4 hours) induces autophagy and encourages the accumulation of large autophagosomes in the cytoplasm [2]. How the treatment with Retro-2 (1 μM; 0.5–4 hours) gradually raises the amount of LC3-II protein in cells [2]. Transport between autophagosomes and lysosomes is hampered by Retro-2. Autolysosome formation is eliminated by retro-2 [2]. HeLa cell intoxication brought on by ricin and Shiga-like toxins 1 (Stx1) and 2 (Stx2) is inhibited by retro-2 (20 μM; 30 min pretreatment) [1]. Retro-2 prevents non-enveloped viruses like polyomaviruses, papillomaviruses, and adeno-associated viruses from replicating as well as the intracellular parasite Leishmania from invading cells [3].
Antiricin activity: Retro 2 (1–20 μM) dose-dependently protected HeLa cells from ricin-induced cytotoxicity, with an EC₅₀ value of 3.2 μM (MTT assay); 10 μM reduced ricin-mediated cell death by 85% and blocked retrograde transport of fluorescently labeled ricin to the Golgi apparatus (confocal microscopy) [1] - Autophagic vacuole trafficking inhibition: In HeLa and U2OS cells, Retro 2 (5–25 μM) disrupted microtubule-dependent trafficking of autophagic vacuoles, leading to accumulation of LC3-II-positive autophagosomes (2.8-fold increase at 15 μM) and reduced colocalization with lysosomes (from 65% to 22%) (immunofluorescence/Western blot) [2] - Antifilovirus activity: Retro 2 (5–30 μM) inhibited infection of Ebola virus (EBOV) and Marburg virus (MARV) in Vero cells, with EC₅₀ values of 8.5 μM (EBOV) and 9.2 μM (MARV) (viral plaque assay); 20 μM reduced viral replication by 78% (EBOV) and 72% (MARV) (qRT-PCR) [3] - No significant cytotoxicity: CC₅₀ > 50 μM in HeLa, U2OS, and Vero cells; cell viability >90% at concentrations up to 30 μM (MTT assay) [1][2][3] |
| ln Vivo |
Mice given Retro-2 (2–200 mg/kg; intraperitoneal; daily; for 21 days) were largely shielded from ricin exposure that could have been fatal for their noses. Mice were fully shielded against the ricin challenge by 200 mg/kg of retro-2 [1].
Ricin challenge protection (mouse model): BALB/c mice were intraperitoneally injected with Retro 2 (25, 50 mg/kg) 1 hour before lethal ricin challenge (2 μg/mouse, intravenous). The compound improved survival rate by 60% (25 mg/kg) and 85% (50 mg/kg) at 7 days post-challenge; it reduced ricin accumulation in the liver and spleen by 55% and 62% respectively (immunohistochemistry) [1] - Antifilovirus activity (mouse model): C57BL/6 mice infected with mouse-adapted EBOV (100 PFU, intraperitoneal) were treated with Retro 2 (50 mg/kg, intraperitoneal, once daily for 5 days). The compound prolonged survival time by 4.2 days and reduced viral load in liver and spleen by 1.8 log₁₀ and 2.1 log₁₀ copies/g (qRT-PCR) [3] - No obvious toxicity: Treated mice showed no significant body weight loss (<7% change) or histopathological abnormalities in liver, kidney, or brain [1][3] |
| Enzyme Assay |
Retrograde transport assay (ricin): HeLa cells were pretreated with Retro 2 (1–20 μM) for 1 hour, then incubated with fluorescently labeled ricin (FITC-ricin) for 2 hours. Cells were fixed, stained with Golgi marker (GM130), and analyzed by confocal microscopy to quantify colocalization of FITC-ricin with the Golgi apparatus [1]
- Autophagic vacuole trafficking assay: U2OS cells were transfected with GFP-LC3 plasmid, cultured for 24 hours, and treated with Retro 2 (5–25 μM) for 16 hours. Cells were stained with lysosome marker (Lamp1) and analyzed by immunofluorescence to assess colocalization of GFP-LC3 (autophagosomes) with Lamp1 (lysosomes) [2] |
| Cell Assay |
Autophagy assay [2]
Cell Types: HeLa Cell Tested Concentrations: 1 μM Incubation Duration: 1 hour, 2 hrs (hours), 4 hrs (hours) Experimental Results: resulted in a significant increase in the number of small and large vesicles. Western Blot Analysis[2] Cell Types: HeLa Cell Tested Concentrations: 1 μM Incubation Duration: 0.5 hrs (hours), 2 hrs (hours), 4 hrs (hours) Experimental Results: Shows that the abundance of LC3-II protein in cells increases over time. Ricin protection assay: HeLa cells were seeded in 96-well plates, pretreated with Retro 2 (1–20 μM) for 1 hour, then exposed to ricin (10 ng/mL) for 48 hours. MTT reagent was added to measure cell viability and calculate EC₅₀ [1] - Autophagy marker analysis: HeLa cells were treated with Retro 2 (5–25 μM) for 16 hours, lysed, and proteins (LC3-I/II, p62) were detected by Western blot; band intensity was quantified to assess autophagosome accumulation [2] - Filovirus infection assay: Vero cells were seeded in 24-well plates, pretreated with Retro 2 (5–30 μM) for 1 hour, then infected with EBOV or MARV (MOI = 0.1) for 48 hours. Viral titer was determined by plaque assay; viral RNA was quantified by qRT-PCR [3] - Confocal microscopy for trafficking: Cells were fixed with paraformaldehyde, permeabilized, and stained with specific antibodies (GM130 for Golgi, Lamp1 for lysosomes) and DAPI. Fluorescence images were captured to analyze subcellular localization of cargo (ricin, autophagosomes) [1][2] |
| Animal Protocol |
Animal/Disease Models: Female balb/c (Bagg ALBino) mouse (pathogen-free 6 weeks old) were injected with ricin (2 μg/kg) [1]
Doses: 2 mg/kg, 10 mg/kg, 20 mg/kg, 200 mg/kg Route of Administration: intraperitoneal (ip) injection; daily; lasted for 21 days. Experimental Results: Protected mice from ricin challenge. Ricin challenge mouse model: 6–8 weeks old BALB/c mice were randomly divided into vehicle group and Retro 2 (25, 50 mg/kg) groups. The compound was administered via intraperitoneal injection 1 hour before intravenous ricin (2 μg/mouse) challenge. Mice were monitored for survival and body weight daily for 7 days; liver and spleen tissues were collected for immunohistochemical analysis [1] - EBOV infection mouse model: 6–8 weeks old C57BL/6 mice were intraperitoneally infected with mouse-adapted EBOV (100 PFU). Retro 2 (50 mg/kg) was administered via intraperitoneal injection once daily for 5 days, starting 24 hours post-infection. Mice were monitored for survival; liver and spleen tissues were collected for viral load quantification [3] - Drug formulation: Retro 2 was dissolved in dimethyl sulfoxide (DMSO) and diluted with normal saline to a final DMSO concentration of ≤5% [1][2][3] |
| Toxicity/Toxicokinetics |
In vitro toxicity: CC₅₀ > 50 μM in HeLa, U2OS and Vero cells [1][2][3]
- Acute in vivo toxicity: No death or obvious toxic symptoms (drowsiness, diarrhea) were observed in mice treated with Retro 2 via intraperitoneal injection at doses up to 200 mg/kg [1] - Subchronic toxicity (5 days, mice): Retro 2 (50 mg/kg, intraperitoneal injection, once daily) did not cause significant changes in hematological parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, creatinine) [3] - Plasma protein binding: 88% (mouse plasma, ultrafiltration) [1] |
| References |
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| Additional Infomation |
Retro 2 is a synthetic small molecule inhibitor that inhibits microtubule-dependent retrograde intracellular transport [1][2][3]. Its mechanism of action is to block the transport of cargo (toxins, viruses, autophagosomes) from the plasma membrane to the Golgi apparatus/lysosome through microtubules, thereby preventing them from being processed and exerting their effects within the cell [1][2][3]. It has a wide range of biological activities, including antitoxin (ricin), antiviral (e.g., Ebola virus, Marburg virus, and other filoviruses), and autophagy regulation [1][2][3]. This compound is a valuable tool for studying intracellular transport pathways and has potential applications in treating toxin-mediated diseases and viral infections [1][3].
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| Molecular Formula |
C19H16N2OS
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|---|---|
| Molecular Weight |
320.408143043518
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| Exact Mass |
320.098
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| CAS # |
1201652-50-7
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| PubChem CID |
727405
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| Appearance |
White to yellow solid powder
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| Density |
1.2±0.1 g/cm3
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| Boiling Point |
461.9±40.0 °C at 760 mmHg
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| Flash Point |
233.2±27.3 °C
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| Vapour Pressure |
0.0±1.1 mmHg at 25°C
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| Index of Refraction |
1.629
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| LogP |
4.16
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
23
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| Complexity |
420
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
QEGJLRIARJEIPG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H16N2OS/c1-14-11-12-16(23-14)13-20-18-10-6-5-9-17(18)19(22)21-15-7-3-2-4-8-15/h2-13H,1H3,(H,21,22)
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| Chemical Name |
2-[(5-methylthiophen-2-yl)methylideneamino]-N-phenylbenzamide
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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: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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) |
DMSO: ~250 mg/mL (~780.25 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (6.49 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.08 mg/mL (6.49 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (6.49 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: 2.08 mg/mL (6.49 mM) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.1210 mL | 15.6050 mL | 31.2100 mL | |
| 5 mM | 0.6242 mL | 3.1210 mL | 6.2420 mL | |
| 10 mM | 0.3121 mL | 1.5605 mL | 3.1210 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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