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Purity: ≥98%
UNC3230 is a selective inhibitor of lipid kinase PIP5K1C. It acts by lowering PIP2 levels in DRG neurons and attenuating hypersensitivity.
| Targets |
UNC3230 targets phosphatidylinositol-4-phosphate 5-kinase type 1C (PIP5K1C) and phosphatidylinositol-5-phosphate 4-kinase, type II, gamma (PIP4K2C).
IC50 for PIP5K1C: ~41 nM (using a microfluidic mobility shift assay). Kd for PIP4K2C: <0.2 μM (using competitive binding assays). At 10 μM, it did not inhibit PIP5K1A, a highly similar family member. [1] |
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| ln Vitro |
When compared to vehicle control, membrane PIP2 levels in dorsal root ganglion (DRG) neurons treated with 100 nM UNC3230 (~2x the IC50) were significantly lower by about 45%. In DRG neurons, UNC3230-cultured cultures considerably inhibited lysophosphatidic acid (LPA)-evoked calcium signaling in comparison to vehicle cultures [1].
UNC3230 reduced membrane Phosphatidylinositol 4,5-bisphosphate (PIP2) levels in cultured dorsal root ganglia (DRG) neurons. Treatment with 100 nM UNC3230 for 20 hours significantly reduced membrane PIP2 levels by approximately 45% compared to vehicle controls, as quantified by average perimeter staining intensity using a PIP2-specific antibody. [1] UNC3230 significantly reduced lysophosphatidic acid (LPA)-evoked calcium signaling in cultured DRG neurons. Pre-incubation for 20 hours with UNC3230 at concentrations of 10 nM, 100 nM, and 1000 nM reduced the area under the curve (AUC) of the calcium response, indicating a blunted signaling effect. [1] |
| ln Vivo |
Two hours after intrathecal injection into wild-type mice, UNC3230 (2 nmol) dramatically enhances the heat-induced paw withdrawal latency of pests, indicating an anti-inflammatory impact [1]. UNC3230 (2 nmol; intrathecal), then co-injecting 1 nmol LPA with UNC3230 (2 nmol, intrathecal) one hour later. When compared to a car, UNC3230 dramatically reduces mechanical allodynia and thermal hyperalgesia [1]. Thermal hyperalgesia and mechanical allodynia are greatly reduced by UNC3230 (2 nmol; intrathecally) [1]. Over the course of many days, the adjuvant (CFA)-inflamed hindpaw showed both mechanical allodynia and thermal hyperalgesia in comparison to the control, but neither the thermal nor the mechanical texture of the non-inflamed hindpaw was impacted [1].
Intrathecal (i.t.) administration of UNC3230 (2 nmol in 20% DMSO) significantly increased noxious heat-evoked paw withdrawal latency in wild-type (WT) mice for up to two hours post-injection, indicating an antinociceptive effect. It did not have an acute effect on mechanical sensitivity. [1] In the LPA-induced neuropathic pain model, i.t. administration of UNC3230 (2 nmol) given one hour before and co-injected with LPA (1 nmol) significantly blunted thermal hyperalgesia and mechanical allodynia compared to vehicle. [1] In the Complete Freund's Adjuvant (CFA) inflammatory pain model, i.t. administration of UNC3230 (2 nmol) two hours before and two hours after CFA injection significantly blunted thermal hyperalgesia and mechanical allodynia in the inflamed hindpaw over a multi-day time course. [1] UNC3230 (2 nmol, i.t.) significantly reduced existing CFA-induced thermal hyperalgesia when administered 48 hours after CFA injection. It did not affect existing mechanical allodynia when administered following CFA inflammation. [1] Peripheral administration of UNC3230 (8 nmol, into the CFA-inflamed hindpaw) reduced thermal hyperalgesia. [1] |
| Enzyme Assay |
The primary enzyme assay was a high-throughput microfluidic mobility shift assay used to screen for inhibitors of recombinant human PIP5K1C. The assay was performed by incubating the N-terminal His6-tagged full-length recombinant human PIP5K1C with or without small molecules in an assay buffer for 10 minutes in 384-well plates. Fluorescein-conjugated substrate [PI(4)P] and ATP at the Km concentration for PIP5K1C (15 μM) were then added and incubated for 40 minutes. Reactions were stopped using EDTA. Fluorescein-conjugated PIP2 and remaining PI(4)P were separated and quantified using the microfluidic mobility shift assay. This assay was used to determine the IC50 of UNC3230 (~41 nM). [1]
Selectivity was assessed using two different assays. The ProfilerPro assay assessed selectivity against 48 kinases. UNC3230 was added to reaction-ready 384-well assay plates containing each kinase in duplicate and incubated for 15 minutes. Matching fluorescent substrates and ATP at the Km for each kinase were added, and the plate was incubated for 90 minutes. Fluorescent substrates and products were separated and quantified using the microfluidic mobility-shift assay. [1] The DiscoveRx KINOMEscan competitive binding assay was used to quantitatively measure interactions between UNC3230 and 100 different kinases. In this assay, UNC3230 and each DNA-tagged kinase were added simultaneously to 384-well plates containing immobilized ligands for each kinase. Plates were incubated for 1 hour, and the amount of kinase bound to the immobilized ligand was quantified using qPCR. This assay determined the Kd values for UNC3230's interaction with various kinases, including a Kd of <0.2 μM for PIP4K2C. [1] |
| Cell Assay |
Dorsal root ganglia (DRG) neurons were cultured from adult mice. To assess PIP2 levels, neurons were treated with vehicle (0.002% DMSO) or 100 nM UNC3230 for 20 hours. Cells were then fixed and immunostained with a PIP2-specific antibody and the neuronal marker NeuN. Confocal images were taken, and the average perimeter (membrane) staining intensity was quantified. [1]
For calcium imaging, cultured DRG neurons were incubated with either vehicle or various concentrations of UNC3230 (10 nM, 100 nM, 1000 nM) for 20 hours. Subsequently, neurons were loaded with the ratiometric calcium indicator dye Fura-2-AM. Calcium responses were measured by stimulating neurons with 10 μM LPA for 90 seconds. The area under the curve (AUC) of the calcium response was quantified for each responding neuron. After LPA stimulation, cultures were washed with HBSS for 120 seconds and then stimulated with 100 mM KCl for 30 seconds to confirm neuron identity. [1] |
| Animal Protocol |
All animal procedures were approved by the Institutional Animal Care and Use Committee. Adult male wild-type mice (6-8 weeks old) were used.
Intrathecal (i.t.) injections (5 μL) were performed in unanesthetized mice using the direct lumbar puncture method. UNC3230 was prepared for i.t. injection at a final concentration of 0.4 mM (2 nmol per 5 μL) in 20% DMSO, or 0.6 mM (3 nmol per 5 μL) in 50% DMSO. [1] Intraperitoneal (i.p.) injections: For i.p. administration, UNC3230 was prepared at 5 mM in 90% corn oil and 10% ethanol and administered at a dose of 20 mg/kg. [1] Intraplantar (into the hindpaw) injections: For peripheral administration, UNC3230 was prepared at a final concentration of 0.4 mM in 20% DMSO and injected into the hindpaw (20 μL per hindpaw; 8 nmol total). [1] LPA-induced neuropathic pain model: UNC3230 (2 nmol, i.t.) or vehicle was administered. One hour later, a second injection of UNC3230 (2 nmol, i.t.) with 1 nmol LPA was administered. [1] CFA inflammatory pain model: UNC3230 (2 nmol, i.t.) or vehicle was administered two hours before and two hours after injecting CFA into one hindpaw. In a separate experiment to test effects on established pain, UNC3230 (2 nmol, i.t.) was administered 48 hours after CFA injection. [1] Thermal sensitivity (Hargreaves assay): A Plantar Test apparatus was used to heat each hindpaw. The latency for hindpaw removal was recorded. The radiant heat source intensity was calibrated so that the average withdrawal latency for WT mice was ~10 seconds, with a cut-off time of 20 seconds. [1] Mechanical sensitivity (von Frey assay): An electronic von Frey apparatus with a semi-flexible tip was used. Three measurements for each hindpaw were taken and averaged to determine the withdrawal threshold in grams. [1] Rotarod assay: Performance on the rotarod was used to assess motor function. UNC3230 (2 nmol, i.t.) did not affect performance. [1] |
| Toxicity/Toxicokinetics |
UNC3230 (2 nmol, i.t.) did not affect performance in the rotarod assay, indicating no acute motor impairment. [1]
An inactive analog of UNC3230 (with a methyl group added to the primary amide) did not exhibit thermal antinociceptive activity, suggesting the observed antinociceptive activity of UNC3230 reflects on-target engagement and not off-target toxicity. [1] |
| References |
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| Additional Infomation |
UNC3230 [5-(cyclohexanecarboxamido)-2-(phenylamino)thiazole-4-carboxamide] was identified from a high-throughput screen of a kinase-focused library (~5,000 compounds) and represents the first reported inhibitor for PIP5K1C and PIP4K2C. [1]
The selectivity profile score (S-score) for UNC3230 was 0.12 (on a scale from 0 to 1.0). Of 38 kinase inhibitors benchmarked in a similar assay, a majority (n=22) were less selective than UNC3230. [1] UNC3230 has a narrow efficacy window and low solubility in appropriate vehicles, which limited the ability to perform full dose-responses in vitro and in vivo. [1] The antinociceptive effects of UNC3230 were similar in magnitude to those observed in Pip5k1c+/- (haploinsufficient) mice. [1] |
| Molecular Formula |
C17H20N4O2S
|
|---|---|
| Molecular Weight |
344.43
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| Exact Mass |
344.13
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| Elemental Analysis |
C, 59.28; H, 5.85; N, 16.27; O, 9.29; S, 9.31
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| CAS # |
1031602-63-7
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| Related CAS # |
1031602-63-7;
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| PubChem CID |
46355372
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| Appearance |
Off-white to light yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.687
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| LogP |
2.49
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
5
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| Heavy Atom Count |
24
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| Complexity |
450
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(N)C=1NC(SC1NC(C2CCCCC2)=O)=NC3=CC=CC=C3
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| InChi Key |
RZCNASHHHSKTGP-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H20N4O2S/c18-14(22)13-16(21-15(23)11-7-3-1-4-8-11)24-17(20-13)19-12-9-5-2-6-10-12/h2,5-6,9-11H,1,3-4,7-8H2,(H2,18,22)(H,19,20)(H,21,23)
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| Chemical Name |
5-(Cyclohexanecarboxamido)-2-(phenylamino)thiazole-4-carboxamide
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| Synonyms |
UNC3230 UNC-3230 UNC 3230
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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 (~362.92 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (6.04 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 20.8 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 | 2.9033 mL | 14.5167 mL | 29.0335 mL | |
| 5 mM | 0.5807 mL | 2.9033 mL | 5.8067 mL | |
| 10 mM | 0.2903 mL | 1.4517 mL | 2.9033 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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