PRN694
目录号 : GC30502
PRN694是一种高度选择且有效的T细胞激酶(ITK)和静息淋巴细胞激酶(RLK)的共价抑制剂，其IC50值分别为0.3和1.4nM。
Cas No.:1575818-46-0
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >99.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Kinase experiment: | Recombinant ITK at a final concentration of 0.5 μM in 50 mM Hepes, pH 7.5, 10 mM MgCl2, 0.01% Triton X-100, and 1 mM EGTA are combined with 1.5 μM PRN694 for 90 min to facilitate binding. The mixture is then diluted 50 fold to initiate dissociating of the ligand from the enzyme, and 10 μL is transferred to a Greiner 384-well black plate. Europium-coupled anti-His6 antibody is added to each well and incubated for 5 min, followed by the addition of an ITK binding fluorescent tracer. The tracer binds to ITK as a function of ligand dissociation, and binding is detected by time-resolved FRET between the europium-coupled antibody and the tracer on a plate reader. Time points acquired are 0.25, 1, 3, 6, and 24 h[1]. |
Cell experiment: | Cells are cultured in vitro at 37°C and 5% CO2 using RPMI 1640 with 10% fetal calf serum. Cells are pretreated for 30 min with PRN694 or other inhibitors and then washed two times. T-cells are then stimulated for 6 h with 1 μg/mL soluble anti-CD3 for CD69 activation, which is detected by flow cytometry, or 45 min with plate-bound anti-CD3 (10 μg/mL plating concentration) and soluble anti-CD28 (1 μg/mL) for downstream signal analysis by immunoblotting. NK cells are stimulated for 6 h with plate-bound anti-CD52 for CD107a/b activation, detected by flow cytometry, or for 45 min for downstream signal analysis by immunoblotting[1]. |
Animal experiment: | Mice are randomized by weight and sensitized with aliquots of 150 μL of 5% oxazolone in 3 parts ethanol and 1 part acetone on their shaved abdomens. Seven days after the sensitization, the mice are challenged with 10 μL of 3% oxazolone on the front and back of the right ears. The left ears are treated with the ethanol/acetone mixture. One hour prior to the challenge, the animals received either vehicle control (5% ethanol, 95% Captex 355 NP/EF, intraperitoneal injection at 5 mL/kg), 20 mg/kg PRN694 in 5% ethanol, 95% Captex (intraperitoneal injection at 5 mL/kg), or 0.5 mg/kg dexamethasone (intraperitoneal injection at 5 mL/kg). A control group of animals receive no oxazolone or drug treatment. Twenty-four hours after the oxazolone challenge, the mice are sacrificed, and a 7 mm disc is punched out of each ear and weighed to measure edema[1]. |
References: [1]. Zhong Y, et al. Targeting interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) using a novel covalent inhibitor PRN694. J Biol Chem. 2015 Mar 6;290(10):5960-78. | |
PRN694 is a highly selective and potent covalent inhibitor of T cell kinase (ITK) and resting lymphocyte kinase (RLK) with IC50s of 0.3 and 1.4 nM, respectively.
PRN694 exhibits high potency against ITK and RLK with IC50 values of 0.3 and 1.4 nM, respectively. With PRN694 pretreatment, CD3-mediated CD69 induction is inhibited both in Jurkat T-cells and freshly isolated primary CD4 or CD8 T-cells. Maximal inhibition of CD69 induction is achieved with PRN694 concentrations ranging from 0.1 to 1.0 μM. Immunoblot analysis of TCR activation pathways reveales that PRN694 blocks activation or nuclear translocation of NFAT1, JunB, pIκBα, and pERK. Results reveal inhibition of Ca2+ signaling with PRN694 at all concentrations above 1 nM. The data show that PRN694 significantly attenuates NK cell FcR-induced killing at concentrations exceeding 0.37 μM. Day 6 flow cytometry analysis reveals that PRN694 significantly inhibits the anti-CD3/CD28-induced proliferation of both CD4 and CD8 T-cells (p<0.01)[1].
The PRN694 occupancy of ITK is 98, 95, and 54% at 1, 6, and 14 h, respectively. The concentrations of PRN694 in the plasma are 2.8, 0.66, and 0.027 μM at 1, 6, and 14 h, respectively. At 14 h, the plasma level of PRN694 is over 10 fold lower than the IC50 in whole blood. RN694 treatment also results in significantly lower weights relative to vehicle (p
[1]. Zhong Y, et al. Targeting interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) using a novel covalent inhibitor PRN694. J Biol Chem. 2015 Mar 6;290(10):5960-78. [2]. Cho HS, et al. A Small Molecule Inhibitor of ITK and RLK Impairs Th1 Differentiation and Prevents Colitis Disease Progression. J Immunol. 2015 Nov 15;195(10):4822-31.
| Cas No. | 1575818-46-0 | SDF | |
| Canonical SMILES | O=C(C1=CC=C(C(F)F)S1)NC2=NC3=CC(CN[C@@H](C)C(C)(C)C)=CC=C3N2C[C@@H]4N(C(C=C)=O)CCC4 | ||
| 分子式 | C28H35F2N5O2S | 分子量 | 543.67 |
| 溶解度 | DMSO : 125 mg/mL (229.92 mM) | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 1.8394 mL | 9.1968 mL | 18.3935 mL |
| 5 mM | 0.3679 mL | 1.8394 mL | 3.6787 mL |
| 10 mM | 0.1839 mL | 0.9197 mL | 1.8394 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
ITK and RLK Inhibitor PRN694 Improves Skin Disease in Two Mouse Models of Psoriasis
The chronic and highly prevalent skin disorder psoriasis vulgaris is characterized by a hyperproliferative epidermis and aberrant immune activity. Many studies have highlighted the role of differentiated T lymphocytes in psoriasis progression. Several biologics are currently available that target proinflammatory cytokines produced by T lymphocytes, but the need for improved therapies persists. The small molecule PRN694 covalently binds ITK and RLK, two Tec kinases activated downstream of T-lymphocyte activation, both of which are up-regulated in psoriatic skin. These Tec kinases are involved in signaling cascades mediating T-lymphocyte proliferation, differentiation, and migration and proinflammatory cytokine production. In vitro analysis showed that PRN694 effectively inhibited IL-17A production from murine T helper type 17-differentiated T lymphocytes. Additionally, PRN694 effectively reduced the psoriasis-like phenotype severity and reduced epidermal proliferation and thickness in both the Rac1V12 and imiquimod mouse models of psoriasis. PRN694 also inhibited CD3+ T-cell and γδ T-cell infiltration into skin regions. Inhibition of ITK and RLK attenuated psoriasis-associated signaling pathways, indicating that PRN694 is an effective psoriasis therapeutic.
Targeting interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK) using a novel covalent inhibitor PRN694
Interleukin-2-inducible T-cell kinase (ITK) and resting lymphocyte kinase (RLK or TXK) are essential mediators of intracellular signaling in both normal and neoplastic T-cells and natural killer (NK) cells. Thus, ITK and RLK inhibitors have therapeutic potential in a number of human autoimmune, inflammatory, and malignant diseases. Here we describe a novel ITK/RLK inhibitor, PRN694, which covalently binds to cysteine residues 442 of ITK and 350 of RLK and blocks kinase activity. Molecular modeling was utilized to design molecules that interact with cysteine while binding to the ATP binding site in the kinase domain. PRN694 exhibits extended target residence time on ITK and RLK and is highly selective for a subset of the TEC kinase family. In vitro cellular assays confirm that PRN694 prevents T-cell receptor- and Fc receptor-induced cellular and molecular activation, inhibits T-cell receptor-induced T-cell proliferation, and blocks proinflammatory cytokine release as well as activation of Th17 cells. Ex vivo assays demonstrate inhibitory activity against T-cell prolymphocytic leukemia cells, and in vivo assays demonstrate durable pharmacodynamic effects on ITK, which reduces an oxazolone-induced delayed type hypersensitivity reaction. These data indicate that PRN694 is a highly selective and potent covalent inhibitor of ITK and RLK, and its extended target residence time enables durable attenuation of effector cells in vitro and in vivo. The results from this study highlight potential applications of this dual inhibitor for the treatment of T-cell- or NK cell-mediated inflammatory, autoimmune, and malignant diseases.
A Small Molecule Inhibitor of ITK and RLK Impairs Th1 Differentiation and Prevents Colitis Disease Progression
In T cells, the Tec kinases IL-2-inducible T cell kinase (ITK) and resting lymphocyte kinase (RLK) are activated by TCR stimulation and are required for optimal downstream signaling. Studies of CD4(+) T cells from Itk(-/-) and Itk(-/-)Rlk(-/-) mice have indicated differential roles of ITK and RLK in Th1, Th2, and Th17 differentiation and cytokine production. However, these findings are confounded by the complex T cell developmental defects in these mice. In this study, we examine the consequences of ITK and RLK inhibition using a highly selective and potent small molecule covalent inhibitor PRN694. In vitro Th polarization experiments indicate that PRN694 is a potent inhibitor of Th1 and Th17 differentiation and cytokine production. Using a T cell adoptive transfer model of colitis, we find that in vivo administration of PRN694 markedly reduces disease progression, T cell infiltration into the intestinal lamina propria, and IFN-γ production by colitogenic CD4(+) T cells. Consistent with these findings, Th1 and Th17 cells differentiated in the presence of PRN694 show reduced P-selectin binding and impaired migration to CXCL11 and CCL20, respectively. Taken together, these data indicate that ITK plus RLK inhibition may have therapeutic potential in Th1-mediated inflammatory diseases.