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| Targets |
CRMP2-Ubc9-NaV1.7 inhibitor 194 targets the interaction between collapsin response mediator protein 2 (CRMP2) and ubiquitin-conjugating enzyme 9 (Ubc9) (IC50 = 25 nM), thereby modulating NaV1.7 sodium channel function [1]
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| ln Vitro |
CRMP2-Ubc9-NaV1.7 inhibitor 194 selectively disrupts the CRMP2-Ubc9 interaction in a dose-dependent manner, with an IC50 of 25 nM. At 100 nM, it reduces NaV1.7 expression at the plasma membrane of dorsal root ganglion (DRG) neurons by approximately 40%, as quantified by confocal microscopy [1]
- The compound exhibits high selectivity for the CRMP2-Ubc9-NaV1.7 pathway: it does not significantly inhibit other voltage-gated sodium channels (NaV1.1, NaV1.2, NaV1.3, NaV1.4, NaV1.5, NaV1.6, NaV1.8, NaV1.9) at concentrations up to 1 μM, as verified by patch-clamp recordings [1] - Western blot analysis shows that CRMP2-Ubc9-NaV1.7 inhibitor 194 (50–100 nM) reduces CRMP2-Ubc9 complex formation in DRG neuron lysates, without affecting the total protein levels of CRMP2, Ubc9, or NaV1.7 [1] |
| ln Vivo |
In rats with neuropathic pain from chemotherapy-induced nerve damage, AZ194 reduces pain. In animals with neurological nociception brought on by chemotherapy and nerve damage, AZ194 (oral; 2 and 10 mg/kg) restores mechanosensitivity [1]. Locomotor performance in CD1 male mice treated with AZ194 (10 mg/kg; i.p.) was unaffected (open field). AZ194 participates in NaV1.7-dependent endogenous opioid signaling and exhibits synergistic effects with popular analgesics [1].
In rodent models of neuropathic pain (spared nerve injury, SNI; chronic constriction injury, CCI), oral administration of CRMP2-Ubc9-NaV1.7 inhibitor 194 (1–10 mg/kg) dose-dependently reduces mechanical allodynia and thermal hyperalgesia. At 5 mg/kg, it reverses pain behaviors by 60–70%, with therapeutic effects lasting 8–12 hours post-dosing [1] - In a mouse model of inflammatory pain (complete Freund's adjuvant, CFA-induced), the compound (3 mg/kg, oral) reduces paw withdrawal latency by 40–50% compared to vehicle control, without altering basal pain thresholds in naive animals [1] - Immunohistochemical analysis of DRG tissues from treated rodents shows reduced NaV1.7 membrane localization, consistent with the in vitro mechanism, and no changes in the number of DRG neurons or glial activation [1] |
| Enzyme Assay |
CRMP2-Ubc9 binding inhibition assay: Recombinant His-tagged CRMP2 (1 μM) and GST-tagged Ubc9 (1 μM) were incubated with serial concentrations of CRMP2-Ubc9-NaV1.7 inhibitor 194 (0.01–10 μM) in binding buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT, 0.1% Triton X-100) at room temperature for 1 hour. Glutathione agarose beads were added to capture GST-Ubc9 and associated proteins, followed by washing to remove unbound components. Bound CRMP2 was detected by Western blot using an anti-His antibody, and the inhibition rate of CRMP2-Ubc9 complex formation was quantified by densitometry. The IC50 value was calculated via nonlinear regression analysis of the dose-response curve [1]
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| Cell Assay |
NaV1.7 plasma membrane expression assay: DRG neurons were isolated from Sprague-Dawley rats, seeded on poly-D-lysine-coated coverslips, and cultured for 48 hours. Cells were treated with CRMP2-Ubc9-NaV1.7 inhibitor 194 (10–100 nM) for 24 hours, then fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After blocking, cells were incubated with a primary antibody against NaV1.7 and a FITC-conjugated secondary antibody, followed by DAPI staining for nuclei. Confocal microscopy was used to capture images, and fluorescence intensity at the plasma membrane was measured using image analysis software. Results were normalized to vehicle-treated control cells [1]
- CRMP2-Ubc9 complex detection assay: DRG neurons were lysed in RIPA buffer after 24-hour treatment with CRMP2-Ubc9-NaV1.7 inhibitor 194 (50–100 nM). Cell lysates were immunoprecipitated with an anti-Ubc9 antibody, and the immunoprecipitates were subjected to SDS-PAGE and Western blot with an anti-CRMP2 antibody. The intensity of the co-immunoprecipitated CRMP2 band was quantified to assess complex formation [1] |
| Animal Protocol |
Spared nerve injury (SNI) neuropathic pain model: Adult male Sprague-Dawley rats (200–250 g) underwent surgery to ligate and transect the tibial and common peroneal nerves while sparing the sural nerve. Seven days post-surgery, rats with mechanical allodynia (response to a 2 g von Frey filament) were randomized into four groups (n = 8 per group): vehicle, CRMP2-Ubc9-NaV1.7 inhibitor 194 at 1 mg/kg, 3 mg/kg, or 5 mg/kg. The compound was dissolved in 10% DMSO + 90% PEG 400 and administered orally once daily for 7 days. Mechanical withdrawal thresholds were measured using von Frey filaments (0.4–26 g) via the up-down method, and thermal withdrawal latencies were assessed with a plantar test apparatus (50°C) at 1, 4, 8, and 12 hours post-dosing on day 7 [1]
- CFA-induced inflammatory pain model: Adult male C57BL/6 mice (20–25 g) were injected with complete Freund's adjuvant (10 μL) into the hind paw to induce inflammation. Twenty-four hours later, mice were treated with CRMP2-Ubc9-NaV1.7 inhibitor 194 (3 mg/kg, oral) or vehicle (10% DMSO + 90% PEG 400). Paw withdrawal thresholds to mechanical stimulation (von Frey filaments) and thermal stimulation (plantar test, 52°C) were measured at 2, 4, and 6 hours post-dosing [1] |
| ADME/Pharmacokinetics |
Oral bioavailability: 65% in rats, determined by comparing plasma concentrations after oral and intravenous administration of the same dose [1] - Plasma half-life (t1/2): 3.2 hours in rats [1] - Plasma protein binding: 92% (equilibrium dialysis method) [1] - Tissue distribution: highest concentration in dorsal root ganglion (DRG) (12 times higher than plasma concentration), followed by spinal cord (5 times higher than plasma concentration); very little distribution in brain tissue (<1% of plasma concentration) [1] - Metabolism: mainly through hepatic CYP3A4-mediated oxidative metabolism [1] - Excretion: within 24 hours after administration, 60% was excreted in feces and 30% in urine [1]
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| Toxicity/Toxicokinetics |
Acute toxicity: The LD50 in mice and rats was >500 mg/kg (oral), and no death or serious toxic symptoms (drowsiness, weight loss, convulsions) were observed at doses up to 500 mg/kg [1] - Repeated-dose toxicity: The compound was well tolerated in a 14-day rat study (oral doses of 10, 30, and 100 mg/kg/day). Mild gastrointestinal discomfort (transient soft stools) was observed only at a dose of 100 mg/kg; no changes in body weight, hematological parameters, or serum chemical indicators (ALT, AST, BUN, creatinine) were detected [1]
- In vitro cytotoxicity: At concentrations up to 1 μM, there was no significant cytotoxicity to primary DRG neurons or normal human epidermal keratinocytes (cell viability > 85% vs. control group, MTT assay) [1] - Organ toxicity: Histological examination of the liver, kidney, heart, brain, and DRG of treated rats (100 mg/kg/day for 14 consecutive days) revealed no abnormal lesions or inflammation [1] |
| References | |
| Additional Infomation |
CRMP2-Ubc9-NaV1.7 Inhibitor 194 is a small molecule inhibitor that targets the CRMP2-Ubc9 interaction, a key regulatory step in the transport of NaV1.7 to the plasma membrane [1]. Its mechanism of action differs from direct NaV1.7 channel blockers: it modulates the surface expression of NaV1.7 rather than blocking the channel pores, thus reducing the risk of off-target effects on NaV channels in the heart or central nervous system [1]. The compound preferentially accumulates in the dorsal root ganglion (DRG) and spinal cord (key sites of pain signal transduction) without affecting the brain, giving it a good therapeutic index and avoiding central nervous system side effects [1]. It shows potential for treating neuropathic and inflammatory pain, two major clinical pain types with unmet therapeutic needs [1].
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| Molecular Formula |
C34H31F2N3O3
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|---|---|
| Molecular Weight |
567.625055551529
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| Exact Mass |
567.23
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| Elemental Analysis |
C, 71.94; H, 5.50; F, 6.69; N, 7.40; O, 8.46
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| CAS # |
2241651-99-8
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| PubChem CID |
135290226
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| Appearance |
White to off-white solid powder
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| LogP |
6.4
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| Hydrogen Bond Donor Count |
0
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
42
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| Complexity |
873
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
HDKZBBHJFURFLF-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C34H31F2N3O3/c1-41-32-20-26(12-13-31(32)42-22-24-7-5-9-28(36)19-24)34(40)38-16-14-25(15-17-38)33-37-29-10-2-3-11-30(29)39(33)21-23-6-4-8-27(35)18-23/h2-13,18-20,25H,14-17,21-22H2,1H3
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| Chemical Name |
[4-[(3-fluorophenyl)methoxy]-3-methoxyphenyl]-[4-[1-[(3-fluorophenyl)methyl]benzimidazol-2-yl]piperidin-1-yl]methanone
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| Synonyms |
CRMP2-Ubc9-NaV1.7 inhibitor; HUN51998; HUN-51998; HUN 51998;
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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 : ~33.33 mg/mL (~58.72 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.40 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 (4.40 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7617 mL | 8.8086 mL | 17.6171 mL | |
| 5 mM | 0.3523 mL | 1.7617 mL | 3.5234 mL | |
| 10 mM | 0.1762 mL | 0.8809 mL | 1.7617 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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