| Size | Price | Stock | Qty |
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| 10mg |
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| Targets |
PROTO-1's primary target is the mechanosensory hair cells of the inner ear, specifically protecting them from aminoglycoside-induced toxicity. The compound acts by preventing aminoglycoside-induced hair cell death, but its precise molecular target and mechanism of action are not fully elucidated. It is known to provide broad protection across a range of neomycin concentrations (25-400 μM) in the zebrafish lateral line assay. Importantly, PROTO-1 does not impair the antibacterial efficacy of aminoglycosides, suggesting that its otoprotective mechanism is selective and does not interfere with the primary antimicrobial action of these drugs. This functional selectivity makes PROTO-1 a valuable tool for mechanistic pathway dissection in ototoxicity research.
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| ln Vitro |
Hair cells are significantly protected by PROTO-1 (1, 10 µM; 1.5 hours) at a wide range of neomycin doses [1].
In vitro studies have demonstrated that PROTO-1 provides significant protection of hair cells over a broad range of neomycin concentrations. In zebrafish lateral line neuromast assays, PROTO-1 shows robust protection against neomycin-induced hair cell death, with an HC50 (half-maximal protective concentration) in the range of 1-10 µM. The compound provides broad protection across neomycin concentrations from 25 to 400 µM. PROTO-1 (1, 10 µM; 1.5 h) treatment shows significant protection of hair cells in a dose-dependent manner. Importantly, PROTO-1 does not interfere with the antibacterial activity of aminoglycosides, making it a valuable tool for studying the selective protection of hair cells without compromising the therapeutic efficacy of these antibiotics. |
| ln Vivo |
In vivo activity of PROTO-1 has been demonstrated in zebrafish models of ototoxicity. The zebrafish lateral line is a well-established model for studying hair cell biology and ototoxicity. In these models, PROTO-1 has been shown to provide reliable and robust protection of mechanosensory hair cells from neomycin-induced damage. The compound's protective effects have been characterized across a broad range of neomycin concentrations, confirming its efficacy in preventing aminoglycoside-induced hair cell death in a living organism. PROTO-1's in vivo activity is further supported by its use as a validated control in high-throughput screening campaigns for otoprotective compounds. However, detailed in vivo studies in mammalian models are not extensively reported.
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| Enzyme Assay |
PROTO-1 is a small-molecule benzothiophene carboxamide. The in vitro enzyme/receptor binding assays for PROTO-1 are not well-defined, as its precise molecular target has not been fully characterized. The compound was identified through high-throughput phenotypic screening for otoprotective agents rather than through target-based screening. Therefore, standard binding assays for specific enzymes or receptors are not applicable. Instead, its activity is assessed through functional cell-based assays that measure hair cell survival. Its mechanism of action is being investigated through studies that examine its effects on cellular pathways involved in aminoglycoside-induced cell death, such as oxidative stress, mitochondrial function, and apoptosis.
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| Cell Assay |
Cell Viability Assay[1]
Cell Types: Sensory Hair Cells Tested Concentrations: 1, 10 µM Incubation Duration: 1.5 hrs (hours) (1 hour pretreatment followed by 0.5 hour exposure to neomycin) Experimental Results: Hair cells were Dramatically affected when exposed to 200 mM Protect neomycin. In vitro cell-based assays for PROTO-1 are conducted using hair cell models, primarily the zebrafish lateral line neuromast system. In a typical assay, zebrafish larvae are exposed to a toxic concentration of neomycin (e.g., 4 mM) in the presence or absence of PROTO-1. After a defined incubation period (e.g., 1.5 hours), the hair cells are stained with a vital dye such as FM1-43 or Yo-Pro-1, and the number of surviving hair cells is quantified by fluorescence microscopy. PROTO-1 is tested at various concentrations (e.g., 1, 10 µM) to determine its protective efficacy. The HC50 value, which represents the concentration that provides 50% protection, is determined from dose-response curves. These assays are used to assess the otoprotective activity of PROTO-1 and to compare its efficacy with other potential otoprotective compounds. |
| Animal Protocol |
In vivo animal experiments for PROTO-1 are typically conducted using zebrafish (Danio rerio) models. The zebrafish lateral line contains mechanosensory hair cells that are structurally and functionally similar to mammalian inner ear hair cells, making it an excellent model for studying ototoxicity and otoprotection. In a typical protocol, zebrafish larvae at 5-6 days post-fertilization are exposed to neomycin (e.g., 4 mM) in the presence or absence of PROTO-1 at various concentrations. After incubation, the larvae are washed and stained with a hair cell-specific dye. The number of surviving hair cells in the neuromasts is then quantified using fluorescence microscopy. PROTO-1's protective effect is measured by comparing hair cell survival in treated versus untreated groups. These experiments are essential for demonstrating the in vivo efficacy of PROTO-1 as an otoprotective agent.
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| ADME/Pharmacokinetics |
PROTO-1 has a molecular weight of 378.88 g/mol and a molecular formula of C17H19ClN4O2S. It is supplied as a solid powder with ≥98% purity. PROTO-1 is soluble in DMSO at 125 mg/mL, supporting concentrated stock solutions for ex vivo mammalian tissue studies. For storage, it is recommended to keep the powder at -20°C for up to 3 years or at 4°C for up to 2 years. In solvent, it can be stored at -80°C for 6 months or at -20°C for 1 month. The compound is stable for short-term storage at 0-4°C for days to weeks. Detailed pharmacokinetic properties, such as half-life, bioavailability, and tissue distribution, have not been extensively characterized.
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| Toxicity/Toxicokinetics |
PROTO-1 is a research compound and is not approved for any clinical or therapeutic use. Its toxicity profile has not been extensively characterized, but it is used as an otoprotective agent in zebrafish models at concentrations that provide significant hair cell protection without apparent toxicity to the larvae. The compound is reported to have reliable and robust protection of hair cells from neomycin, suggesting a favorable safety profile in the context of its research applications. However, comprehensive toxicological data, including acute and chronic toxicity studies, are not available. As with all research chemicals, standard laboratory safety precautions should be followed when handling PROTO-1.
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| References |
[1]. Owens KN, et al. Identification of genetic and chemicalm modulators of zebrafish mechanosensory hair cell death. PLoS Genet. 2008 Feb 29;4(2):e1000020.
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| Additional Infomation |
PROTO-1 is a research-use-only compound and is not approved for any clinical or therapeutic use. It is a small-molecule benzothiophene carboxamide identified through high-throughput screening as an otoprotective agent that prevents aminoglycoside-induced hair cell death. PROTO-1 is used as a tool compound in studies of ototoxicity-induced hair cell death, particularly in the zebrafish lateral line model. It provides reliable and robust protection of hair cells from neomycin without interfering with the antibacterial activity of aminoglycosides. The compound is supplied as a solid with ≥98% purity and is intended for research use only (RUO). PROTO-1 is a valuable tool for studying the mechanisms of aminoglycoside ototoxicity and for screening potential otoprotective agents.
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| Molecular Formula |
C17H19CLN4O2S
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|---|---|
| Molecular Weight |
378.88
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| Exact Mass |
378.091
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| CAS # |
312951-85-2
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| PubChem CID |
1069493
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
430.9±45.0 °C at 760 mmHg
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| Flash Point |
214.4±28.7 °C
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| Vapour Pressure |
0.0±1.0 mmHg at 25°C
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| Index of Refraction |
1.698
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| LogP |
4.3
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
25
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| Complexity |
501
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C([H])=C([H])C(=C([H])C=1[H])N([H])C(N([H])C1=C(C(N([H])[H])=O)C2=C(C([H])([H])N(C([H])([H])C([H])([H])[H])C([H])([H])C2([H])[H])S1)=O
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| InChi Key |
QBLKUVUKIZEWAL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H19ClN4O2S/c1-2-22-8-7-12-13(9-22)25-16(14(12)15(19)23)21-17(24)20-11-5-3-10(18)4-6-11/h3-6H,2,7-9H2,1H3,(H2,19,23)(H2,20,21,24)
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| Chemical Name |
2-[(4-chlorophenyl)carbamoylamino]-6-ethyl-5,7-dihydro-4H-thieno[2,3-c]pyridine-3-carboxamide
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| Synonyms |
PROTO-1 PROTO 1 PROTO1
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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) |
DMSO : ~125 mg/mL (~329.92 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.49 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 (5.49 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.6394 mL | 13.1968 mL | 26.3936 mL | |
| 5 mM | 0.5279 mL | 2.6394 mL | 5.2787 mL | |
| 10 mM | 0.2639 mL | 1.3197 mL | 2.6394 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
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