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| Targets |
SPR inhibitor 3 targets sepiapterin reductase (SPR), a key enzyme in the tetrahydrobiopterin (BH4) biosynthesis pathway. BH4 is an essential cofactor for several enzymes, including nitric oxide synthase (NOS), phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase. Dysregulation of BH4 levels has been implicated in various pathological conditions, including neuropathic pain, inflammatory pain, Parkinson's disease, and psychiatric disorders. By inhibiting SPR, the compound reduces BH4 levels, thereby modulating the activity of BH4-dependent enzymes. SPR inhibitor 3 is a class of small-molecule compounds used to inhibit the activity of sepiapterin reductase.
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| ln Vitro |
SPR inhibitor 3 (SPRi3) also considerably lowers SPR activity in primary cultures of mouse sensory neurons (IC50=0.45 μM) without influencing the activity of GTP ring hydroxylase 1 enzyme (GCH1) [1] .
SPR inhibitor 3 demonstrates potent in vitro activity as a sepiapterin reductase inhibitor. The compound displays high binding affinity to human SPR in cell-free assays with an IC50 of 74 nM. In cell-based assays, SPR inhibitor 3 efficiently reduces biopterin levels with an IC50 of 5.2 microM. The compound's activity is concentration-dependent, with potent inhibition observed at nanomolar to micromolar concentrations. SPR inhibitor 3 is widely studied in neurological disease models, including Parkinson's disease, neuropathic pain, and psychiatric disorders, where BH4 dysregulation plays a role. Its potency and selectivity make it a valuable tool for studying BH4 metabolism and its role in various diseases. |
| ln Vivo |
In vivo, SPR inhibitor 3 has demonstrated efficacy in reducing neuropathic and inflammatory pain through a reduction of BH4 levels. The compound is studied in neurological disease models where BH4 dysregulation plays a role. By inhibiting SPR and reducing BH4 levels, the compound modulates the activity of BH4-dependent enzymes, including nitric oxide synthase, which is involved in pain signaling. The compound's in vivo activity has been characterized in various animal models of pain and neurological disorders. Its ability to cross the blood-brain barrier is a key consideration for its central nervous system effects. SPR inhibitor 3 is valuable for investigating metabolic and neurological pathways and exploring novel therapeutic strategies targeting SPR-related mechanisms.
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| Enzyme Assay |
In vitro enzyme assays for SPR inhibitor 3 involve measuring the inhibition of sepiapterin reductase activity. The enzyme is incubated with varying concentrations of the test compound, a substrate (sepiapterin), and the cofactor NADPH. The production of the reduced product (tetrahydrobiopterin or dihydrobiopterin) is quantified by HPLC or fluorometric detection. IC50 values are calculated by plotting percent inhibition against compound concentration using non-linear regression analysis. The compound displays high binding affinity to human SPR in cell-free assays with an IC50 of 74 nM. Selectivity assays compare the compound's activity against related enzymes in the BH4 biosynthesis pathway. The assay is performed in appropriate buffer conditions with controls for non-specific inhibition. Each concentration is typically tested in duplicate or triplicate.
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| Cell Assay |
In vitro cellular assays for SPR inhibitor 3 are performed using cells that express sepiapterin reductase and produce biopterins. Cells are treated with varying concentrations of the compound for defined time periods. Biopterin levels (including tetrahydrobiopterin, dihydrobiopterin, and biopterin) are measured in cell lysates using HPLC with fluorescence detection or LC-MS/MS. The compound efficiently reduces biopterin levels in cell-based assays with an IC50 of 5.2 microM. Cytotoxicity is assessed in parallel using standard viability assays to ensure that observed effects are not due to cell death. The compound's effects on downstream BH4-dependent enzyme activities can also be assessed. Results are expressed as percent reduction in biopterin levels compared to vehicle-treated controls.
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| Animal Protocol |
In vivo animal studies for SPR inhibitor 3 are conducted using rodent models of pain and neurological disorders. The compound is administered via oral gavage, intraperitoneal injection, or subcutaneous injection at various doses and schedules. Pain behaviors are assessed using models of neuropathic pain (spared nerve injury, chronic constriction injury) and inflammatory pain (complete Freund's adjuvant, formalin test). Mechanical allodynia and thermal hyperalgesia are measured using von Frey filaments and Hargreaves apparatus, respectively. BH4 levels are measured in target tissues (spinal cord, dorsal root ganglia, brain) to confirm target engagement. Pharmacokinetic studies assess drug concentrations in plasma and tissues. Body weight and clinical observations are monitored as safety indicators. Efficacy is expressed as reversal of pain behaviors compared to vehicle-treated controls.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of SPR inhibitor 3 have been characterized in preclinical studies. The compound has a molecular formula of C14H18N2O3 and a molecular weight of 262.30 g/mol. It is soluble in DMSO and other organic solvents. The compound is designed to be orally bioavailable for convenient dosing in research settings. Comprehensive pharmacokinetic parameters including half-life, volume of distribution, clearance, and oral bioavailability have been characterized in animal models. The compound's ability to cross the blood-brain barrier is relevant for its effects on central nervous system BH4 levels. Its pharmacokinetic profile supports its use in preclinical studies of pain and neurological disorders. Detailed pharmacokinetic data are available from research publications.
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| Toxicity/Toxicokinetics |
SPR inhibitor 3 is intended for laboratory research use only and has not undergone comprehensive clinical toxicology testing. As an inhibitor of BH4 biosynthesis, the compound would be expected to have effects on nitric oxide production and neurotransmitter synthesis, which could have various physiological consequences. Standard in vitro cytotoxicity assays in cell lines are typically performed alongside efficacy studies to rule out nonspecific toxicity. In vivo, animals are monitored for signs of toxicity including body weight changes, behavioral abnormalities, and clinical observations. Comprehensive toxicological characterization including genotoxicity, cardiotoxicity, and repeated-dose toxicity studies has not been reported in the public domain. The compound is not approved for human use and is strictly intended for research purposes.
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| References |
[1]. Latremoliere A, et al. Reduction of Neuropathic and Inflammatory Pain through Inhibition of the Tetrahydrobiopterin Pathway. Neuron. 2015 Jun 17;86(6):1393-406.
[2]. Cronin SJF, et al. The metabolite BH4 controls T cell proliferation in autoimmunity and cancer. Nature. 2018 Nov;563(7732):564-568. [3]. Haruki H, et al. Tetrahydrobiopterin Biosynthesis as a Potential Target of the Kynurenine Pathway Metabolite Xanthurenic Acid. J Biol Chem. 2016 Jan 8;291(2):652-7. |
| Additional Infomation |
SPR inhibitor 3 (SPRi3) is a potent inhibitor of sepiapterin reductase (SPR) with an IC50 of 74 nM in cell-free assays and 5.2 microM in cell-based assays. It decreases neuropathic and inflammatory pain through a reduction of BH4 levels. The compound has a molecular formula of C14H18N2O3 and a molecular weight of 262.30 g/mol. SPR inhibitor 3 is studied in neurological disease models including Parkinson's disease, neuropathic pain, and psychiatric disorders. It is valuable for investigating metabolic and neurological pathways and exploring novel therapeutic strategies targeting SPR-related mechanisms. The compound has not entered clinical trials and is available for research purposes only.
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| Molecular Formula |
C14H18N2O3
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| Molecular Weight |
262.3043
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| Exact Mass |
262.131
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| CAS # |
1292285-54-1
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| PubChem CID |
52911386
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| Appearance |
Off-white to gray solid powder
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| LogP |
1.6
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
19
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| Complexity |
311
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CC1=C(C2=C(N1)C=CC(=C2)O)CCNC(=O)COC
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| InChi Key |
YBXBWBBVLXZQBJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C14H18N2O3/c1-9-11(5-6-15-14(18)8-19-2)12-7-10(17)3-4-13(12)16-9/h3-4,7,16-17H,5-6,8H2,1-2H3,(H,15,18)
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| Chemical Name |
N-[2-(5-hydroxy-2-methyl-1H-indol-3-yl)ethyl]-2-methoxyacetamide
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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 Vitro) |
DMSO : ~100 mg/mL (~381.24 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (9.53 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 25.0 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: ≥ 2.5 mg/mL (9.53 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 25.0 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: ≥ 2.5 mg/mL (9.53 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.8124 mL | 19.0621 mL | 38.1243 mL | |
| 5 mM | 0.7625 mL | 3.8124 mL | 7.6249 mL | |
| 10 mM | 0.3812 mL | 1.9062 mL | 3.8124 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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