c-Jun N-terminal kinase (JNK). SU3327 (compound 9) is a selective JNK inhibitor that prevents protein-protein interactions between JNK and JNK Interacting Protein (JIP), acting as a substrate-competitive inhibitor that binds to the docking site of the kinase. [1, 2]
In a kinase inhibition assay, SU3327 showed an IC50 of 0.7 μM. In a DELFIA displacement assay measuring displacement of biotinylated pepJIP1 from GST-JNK1, the IC50 was 239 nM. [1]
SU3327 is highly selective, showing >100 μM IC50 against p38α (a closely related MAPK) and only 11% inhibition of Akt at 100 μM. [1]
c-Jun N-terminal kinase (JNK). SU3327 is a selective JNK inhibitor that prevents protein-protein interactions between JNK and JNK Interacting Protein (JIP), acting as a substrate-competitive inhibitor that binds to the docking site of the kinase. [2]

In a cell-based LanthaScreen kinase assay using HeLa cells stably expressing GFP-c-Jun (1-79), SU3327 inhibited TNF-α-stimulated phosphorylation of c-Jun with an EC50 of 6.23 μM. [1]
In hCMEC/D3 human cerebral microvascular endothelial cells, SU3327 (25 nM) pretreatment significantly reduced LPS-induced PMN rolling and adhesion. It also suppressed LPS-induced activation of AP-1 (as measured by c-Jun Ser63 phosphorylation) and reduced VCAM-1 expression, but did not affect E-selectin or ICAM-1 expression. [3]

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In HeLa cells (EC50=6.23 μM), SU3327 (Compound 9) can prevent TNF-α-stimulated c-Jun phosphorylation[1]. Polymorphonuclear leukocyte (PMN) rolling and attachment to human brain microvascular endothelial cells (hCMEC/D3) is efficiently reduced by SU3327 (25 nM). It also inhibits AP-1 activation and drastically lowers VCAM-1 expression[3].

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In primary human astrocytes (HA) grown on flexible culture membranes, pre-treatment with SU3327 (25 μM for 10 minutes) significantly reduced traumatic injury-induced (55% membrane deformation) HA monolayer retraction. Pre-treatment with SU3327 also reduced JNK1/2 phosphorylation in HA following traumatic injury. [2]

In insulin-resistant db/db mice (11-week-old male BKS.Cg-+Leprᵈᵇ/+Leprᵈᵇ/OlaHsd), intraperitoneal administration of SU3327 (as compound 9) at 25 mg/kg significantly reduced blood glucose levels following insulin challenge compared to vehicle control. Mice were fasted 6 hours before drug administration, followed 30 minutes later by bovine insulin (0.75 mg/kg i.p.). Blood glucose was measured at designated time points. [1]

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In diabetic electrode models, SU3327 (Compound 9; 25 mg/kg; intraperitoneal injection; cosmetic BKS.Cg-+Leprdb/+Leprdb/+Leprdb/OlaHsd db/db electrode) can recuperate the insulin electrodes' processing capacity [1].

In Vitro Kinase Assay (LanthaScreen): The time-resolved fluorescence resonance energy transfer (TR-FRET) assay was performed in 384-well plates. Each well received JNK (35 ng/mL), ATF2 (400 nM), and ATP (200 μM) in 50 mM HEPES, 10 mM MgCl₂, 1 mM EGTA, 0.01% Brij-35, pH 7.5, and test compounds. The kinase reaction was performed at room temperature for 1 hour. After addition of terbium-labeled antibody and EDTA, the signal was measured at 520/495 nm emission ratio on a fluorescence plate reader. [1]
DELFIA Assay (pepJIP1 Displacement): To each well of 96-well streptavidin-coated plates, 100 μL of a 100 ng/mL solution of biotin-labeled pep-JIP1 (Biotin-lc-KRPKRPITLNLF) was added. After 1 hour incubation, unbound biotin-pep-JIP1 was removed by washing. Then, 87 μL of Eu-labeled anti-GST antibody solution (300 ng/mL), 2.5 μL DMSO containing test compound, and 10 μL of GST-JNK1 (final 10 nM) were added. After 1 hour incubation at 0°C, wells were washed to remove unbound protein. Enhancement solution was added, and fluorescence was measured (excitation 340 nm, emission 615 nm). [1]

Cell-Based c-Jun Phosphorylation Assay (LanthaScreen): HeLa cells stably expressing GFP-c-Jun (1-79) were plated in white 384-well plates at 10,000 cells/well in 32 μL assay medium. After overnight incubation, cells were pretreated with SU3327 for 60 minutes, then stimulated with TNF-α (2 ng/mL) for 30 minutes. Cells were lysed in buffer containing terbium-labeled anti-phospho-c-Jun (pSer73) detection antibody. After 1 hour equilibration, TR-FRET emission ratios were determined (excitation 340 nm, emission 520/490 nm). [1]
PMN Rolling/Adhesion Assay: hCMEC/D3 cells were grown in parallel flow perfusion slides. Confluent monolayers were pretreated with SU3327 (25 nM) for 30 minutes, then stimulated with LPS (1 μg/mL) for 6 hours. Human PMN (1×10⁶/mL) were perfused over the endothelial monolayers at 0.7 dyn/cm² laminar shear stress for 5 minutes. PMN rolling/adhesion were video-recorded and quantified. [3]
qPCR for Adhesion Molecules: hCMEC/D3 cells were pretreated with SU3327 (25 nM) for 30 minutes, then stimulated with LPS (1 μg/mL) for 6 hours. Total RNA was extracted using TRIzol, and cDNA was synthesized. Expression of E-selectin, ICAM-1, and VCAM-1 was assessed using TaqMan Gene Expression Assays. [3]
ELISA for AP-1 and NF-κB Activation: hCMEC/D3 cells were pretreated with SU3327 (25 nM) for 30 minutes, then stimulated with LPS (1 μg/mL) for 30 minutes (AP-1) or 1 hour (NF-κB). PathScan Phospho-c-Jun (Ser63) and Phospho-NF-κB p65 (Ser536) Sandwich ELISA Kits were used according to manufacturer's instructions. [3]

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Human Astrocyte (HA) Culture and Traumatic Injury: Primary human astrocytes were grown on fibronectin-coated Bioflex culture plates in VascuLife medium. Confluent HA monolayers were pre-treated with SU3327 (25 μM) for 10 minutes prior to traumatic injury. Traumatic injury was delivered using the Cell Injury Controller II, which applies a 50-ms burst of gas to deform the flexible membrane, producing 55% membrane deformation (4.0 peak PSI) to mimic severe blunt trauma. HA monolayer retraction was assessed 30 minutes post-injury by measuring the surface area covered by fluorescently stained HA (CFDA) relative to the dark background using ImageJ software. [2]
Western Blotting for JNK Phosphorylation: HA were lysed in hot SDS sample buffer 30 minutes after injury. Proteins were separated by 12% SDS-PAGE, transferred to PVDF membranes, and probed with rabbit polyclonal antibody against phospho-JNK1/2 (phos T183 and Y185, detecting bands at 49 and 55 kDa). Total JNK1/2 (detecting bands at 48 kDa) and GAPDH (36 kDa) were used as loading controls. Band intensities were captured and quantified. Pre-treatment with SU3327 significantly reduced JNK1/2 phosphorylation following traumatic injury. [2]

Animal/Disease Models: Male BKS.Cg-+Leprdb/+Leprdb/OlaHsd db/db mice (11 weeks old) were injected with insulin [1]. ]
Doses: 25 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: Resulted in a statistically significant reduction in blood glucose levels.

Liquid chromatography/mass spectrometry bioavailability analysis demonstrated that SU3327 (compound 9) has favorable microsomal and plasma stability with a half-life (T₁/₂) of 27 minutes, supporting its use in further in vivo experiments. [1]

[1]. Design, synthesis, and structure-activity relationship of substrate competitive, selective, and in vivo active triazole and thiadiazole inhibitors of the c-Jun N-terminal kinase. J Med Chem. 2009 Apr 9;52(7):1943-52.

[2]. Traumatic injury elicits JNK-mediated human astrocyte retraction in vitro. Neuroscience. 2014 Aug 22;274:1-10.

[3]. Pretreatment of human cerebrovascular endothelial cells with CO-releasing molecule-3 interferes with JNK/AP-1 signaling and suppresses LPS-induced proadhesive phenotype. Microcirculation. 2015 Jan;22(1):28-36.

Halicin is a thiadiazole compound with the structure 1,3,4-thiadiazole-2-amine, where the 5-position is substituted with a (5-nitro-1,3-thiazol-2-yl)thiodiacyl group. It is a c-Jun N-terminal kinase inhibitor (IC50 = 0.7 μM) and possesses antibacterial activity. It is both a c-Jun N-terminal kinase inhibitor and an antibacterial agent. Halicin belongs to the thiadiazole class, 1,3-thiaazole class, primary amino compounds, C-nitro compounds, and organosulfur compounds.

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SU3327 (compound 9) is a 5-(5-nitrothiazol-2-ylthio)-1,3,4-thiadiazol-2-amine derivative. It was identified through comprehensive structure-activity relationship studies on a novel series of JNK inhibitors that target the JIP-JNK interaction site (substrate-competitive inhibitors). Unlike ATP-competitive inhibitors, SU3327 binds to the docking site of the kinase, preventing protein-protein interactions between JNK and JIP. [1]
Modeling studies suggest that SU3327 binds at the JIP site with the nitrothiazol group crossing the ridge close to residues Arg127 and Cys163, forming a hydrogen bond with Arg127. [1]
SU3327 is a selective JNK inhibitor that prevents protein-protein interactions between JNK and JNK Interacting Protein (JIP). It has been used to demonstrate that JNK activation mediates human astrocyte retraction following traumatic injury and LPS-induced cerebrovascular endothelial cell activation. [2, 3]

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SU3327 is a selective JNK inhibitor that prevents protein-protein interactions between JNK and JNK Interacting Protein (JIP), a scaffold protein responsible for regulating JNK signaling. Unlike SP600125 (an ATP-competitive inhibitor), SU3327 is a substrate-competitive inhibitor that binds to the docking site of the kinase. [2]
This study demonstrated that traumatic injury induces JNK-mediated human astrocyte retraction, and that SU3327 pre-treatment reduces both JNK phosphorylation and injury-induced astrocyte retraction. The authors suggest that JNK inhibition could be a potential therapeutic approach for traumatic brain injury (TBI). [2]

C₅H₃N₅O₂S₃

261.30

260.945

C, 22.98; H, 1.16; N, 26.80; O, 12.25; S, 36.81

40045-50-9

11837140

Light yellow to yellow solid powder

1.888g/cm3

549.841ºC at 760 mmHg

160 °C(dec.)

286.334ºC

1.793

2.089

1

9

2

15

251

0

NQQBNZBOOHHVQP-UHFFFAOYSA-N

InChI=1S/C5H3N5O2S3/c6-3-8-9-5(14-3)15-4-7-1-2(13-4)10(11)12/h1H,(H2,6,8)

5-[(5-nitro-1,3-thiazol-2-yl)sulfanyl]-1,3,4-thiadiazol-2-amine

SU-3327; Halicin; 5-[(5-nitro-1,3-thiazol-2-yl)sulfanyl]-1,3,4-thiadiazol-2-amine; 5-((5-Nitro-1,3-thiazol-2-yl)sulfanyl)-1,3,4-thiadiazol-2-amine; RefChem:783357; 40045-50-9; SU3327; compound 9; SU 3327

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~62.5 mg/mL (~239.19 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.96 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.96 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.

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 (Please use freshly prepared in vivo formulations for optimal results.)