APY0201

PIKfyve (IC50 = 5.2 nM); IL-23

APY0201 在人 PBMC 中以 99 nM 抑制 IL-12p40。 APY0201 对 IL-12p70 和 IL-12p40 的产生比 TNF-α 表现出显着的选择性，并且这种选择性在不同物种之间得以维持。APY0201 的作用与抗 IL-12/23 抗体不同，它通过抑制这些促炎细胞因子的产生来起作用与其他细胞因子（包括肿瘤坏死因子-α (TNF-α)）相比具有特征选择性。

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APY0201显示出对IL-12/23产生的选择性抑制。 APY0201与结合ArPIKFyve的蛋白质复合物结合。 APY0201被鉴定为PIKfyve激酶的ATP竞争性抑制剂[1]。

口服 30 mg/kg 剂量的 APY0201 显示 IL-12p70 产生显着减少（相对于载体对照抑制 78%），这意味着 APY0201 对 IL-12 的抑制潜力在动物实验中得到证实。 APY0201改善结肠炎实验模型中的炎症[1]
为了确定PIFKfyve抑制对体内炎症反应的影响，我们在IL-10敲除（KO）CD4+T细胞过继转移诱导的炎症性肠病（IBD）小鼠模型中评估了APY0201。46，47在该模型中，从患病的IL-10缺陷小鼠中收集脾细胞和肠系膜淋巴结细胞，纯化高达95%的CD4+T细胞，将其转移到雌性SCID小鼠体内，在3周内诱导自发性结肠炎。通过湿结肠重量评估结肠炎的严重程度。对大便硬度进行评分，以评估APY0201对结肠炎的治疗效果。在第21天处死动物，并测量结肠重量。与正常对照组相比，结肠炎赋形剂对照组的结肠重量比有所增加。APY0201的每日给药以剂量依赖的方式显著减少了结肠重量的增加（图7a；3、10和30 mg/kg的剂量分别减少了19.7%、25.4%和73.3%），30 mg/kg的APY02001的效果与15 mg/kg B.I.D.的泼尼松龙（PSL）的效果相当（图7b，减少了81.0%）。粪便稠度检查显示，赋形剂对照组在处死当天出现严重腹泻，而APY0201以剂量依赖的方式显著预防了腹泻的发展（图7c和d）。每天两次服用PSL可以改善结肠炎小鼠模型的炎症。然而，与赋形剂或正常对照组相比，PSL治疗导致慢性给药后体重显著下降，尽管该药物缓解了腹泻并减轻了结肠重量（请参阅补充信息）。相反，与正常对照组相比，服用APY0201不会导致体重差异。这些结果清楚地表明，APY0201是口服的，在通过过继转移IL-10 KO CD4+T细胞诱导的结肠炎小鼠模型中显著有效，没有显示出任何不良反应的迹象。

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靶点识别[1]
将带有FLAG肽表位（7-10）的诱饵化合物与IFNγ/SAC刺激的小鼠TG-PEC制备的细胞裂解物一起孵育。用珠结合的抗FLAG抗体对诱饵化合物-蛋白质复合物进行免疫沉淀，然后用Lys-C内切蛋白酶消化诱饵化合物相关蛋白。如前所述，使用DNLC–MS/MS系统分析所得肽。23,24重复分析四次，至少两次不同分析的观察峰在补充信息的表2中和表S2中表示为“发现”。实验的详细程序见补充信息。

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PIKfyve同源性建模与对接研究[1]
PIP4KIIβ（PI4K2B，PDB：1BO1）的已知晶体结构被用作分子操作环境（MOE）软件的模板，以产生PIKfyve激酶的同源模型。PIP4KIIβ与PIKfyve激酶具有最接近的序列同一性。MOE软件包中的对接功能用于将APY0201对接到获得的PIKfyve同源模型中。详细程序见补充信息。

将小鼠 TG-PEC 或人 PBMC 在 100 ng/mL 小鼠或人 IFN-γ 和 0.05%w/v 金黄色葡萄球菌 Cowan I 菌株 (SAC ）[1]。

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细胞分离[1]
如补充信息所述，从雌性BALB/c小鼠（6周）收集小鼠TG-PEC细胞。如补充信息所述，从健康志愿者的外周血中分离出人PBMC。

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细胞刺激[1]
如补充信息所述，在100ng/mL小鼠或人IFN-γ和0.05%w/v SAC的存在下，将小鼠TG-PEC或人PBMC与受试化合物APY0201 一起孵育。

Mice: Female BALB/c mice (n=3) are used. Systemic or portal vein blood samples taken while sedated. Mice are given diethyl ether anesthesia after 30 minutes, and blood samples are obtained by cardiac puncture. Blood is drawn into tubes containing a 0.5 M-EDTA solution (pH 8.0).

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Murine IL-10−/− cell transfer colitis [1]
Colitis was induced in female SCID mice (n = 8) by adoptive transfer of spleen and mesenteric lymph node cells from diseased IL-10−/− mice, as described previously. The tested compound APY0201 was administered from day 0 to mice with experimental colitis. The mice were sacrificed for assessing inflammation 21 days after cell transfer. The severity of colitis was assessed according to wet colon weight. Scoring of stool consistency was performed once in 2 days (0: normal beaded stool, 2: soft stool, 4: diarrhea). The detailed experimental procedure is provided in the Supplementary information.

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PK profile [1]
Female BALB/c mice (n = 3) were used. For intravenous administration, the tested compound was dissolved in 80% PEG 400/20% water (3 or 10 mg/mL/kg) for a dose of 3 or 10 mg/kg, respectively. For oral administration, the tested compound APY0201 was suspended in 0.5% methyl cellulose (30 mg/5 mL/kg). Blood samples were collected at designated time points by cardiac puncture (systemic) or from the portal vein (portal) under anesthesia. The detailed procedures for preprocessing, analysis, and calculation of the PK parameters are described in the Supplementary information.

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Mouse whole blood ex vivo assay [1]
The vehicle (0.5% methyl cellulose) and tested compound APY0201 were orally administered to female BALB/c mice (6 weeks). After 30 min, the mice were anesthetized, and blood samples were collected by cardiac puncture. The detailed procedures for the preprocessing and analysis are described in the Supplementary information.

The PK parameters obtained after intravenous administration at 3 or 10 mg/kg and oral dosing at 30 mg/kg are shown in Figure 6. For intravenous administration, the PK parameters of initial drug concentration (C0) and area under the curve to time infinity (AUCinf) showed good linearity between the two doses, and APY0201 showed low total body clearance (CLtot = 1.0 L/h/kg) (Fig. 6a). After oral administration, heart and portal plasma concentrations were determined to assess the first-pass effect (Fig. 6b). The maximum APY0201 concentration (Cmax) value in the heart was 7.2 μM, and the plasma concentration was 3.5 μM at 6 h. The apparent fraction absorbed and availability at the liver were estimated to be 68% and 76% of the dose, respectively, which indicated a good bioavailability of 52% (Table 3). Because good bioavailability with oral administration was obtained, APY0201 was examined for its oral therapeutic efficacy for once-daily dosing at 30 mg/kg.[1]

[1]. Structure-activity relationship study, target identification, and pharmacological characterization of a small molecular IL-12/23 inhibitor, APY0201. Bioorg Med Chem. 2014 Jun 1;22(11):3021-9.

Interleukin-12 (IL-12) and IL-23 are proinflammatory cytokines and therapeutic targets for inflammatory and autoimmune diseases, including inflammatory bowel diseases, psoriasis, rheumatoid arthritis, and multiple sclerosis. We describe the discovery of APY0201, a unique small molecular IL-12/23 production inhibitor, from activated macrophages and monocytes, and demonstrate ameliorated inflammation in an experimental model of colitis. Through a chemical proteomics approach using a highly sensitive direct nanoflow LC-MS/MS system and bait compounds equipped with the FLAG epitope associated regulator of PIKfyve (ArPIKfyve) was detected. Further study identified its associated protein phosphoinositide kinase, FYVE finger-containing (PIKfyve), as the target protein of APY0201, which was characterized as a potent, highly selective, ATP-competitive PIKfyve inhibitor that interrupts the conversion of phosphatidylinositol 3-phosphate (PtdIns3P) to PtdIns(3,5)P2. These results elucidate the function of PIKfyve kinase in the IL-12/23 production pathway and in IL-12/23-driven inflammatory disease pathologies to provide a compelling rationale for targeting PIKfyve kinase in inflammatory and autoimmune diseases.[1]

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APY0201 is an orally-available PIKfyve kinase inhibitor that is selective for all tested kinases, GPCRs, ion channels, and enzymes, and it is a powerful tool for understanding the role of PIKfyve and PtdIns(3,5)P2 in immunological and inflammatory responses. The administration of APY0201 can be used to assess PIKfyve function in immune cells and animals with normal architecture of the PIKfyve–ArPIKfyve–Sac3 complex. Exposure to APY0201 in vitro blocked production of IL-12/23, which highlighted the effect of selective inhibition of this kinase. The data presented here indicate a unique role of PIKfyve kinase in cytokine production. Because PIKfyve inhibition blocks production of IL-12 and IL-23 from activated macrophages, APY0201 may control the cytokine modulation function of these cell types and reduce the pathological proinflammatory cytokines IL-12/23 with negligible influence on other cytokines, including TNF-α. Moreover, the therapeutic impact of PIKfyve inhibition in a mouse model of IBD included reduction in inflammation without affecting body weight. In the in vitro assay using mouse whole blood for IL-12p70-inhibitory activity, APY0201 demonstrated potent inhibitory activity against IL-12p70 production with the IC50 value of 880 nM. At the therapeutic dose (30 mg/kg daily), the mean Cmax was 7.2 μM, which is sufficient to inhibit IL-12p70 production in whole blood almost completely, and the estimated plasma concentration at 12 h is still close to 880 nM, which indicated that an appropriate amount of APY0201 was maintained to inhibit IL-12p70 production for nearly a half day (Fig. 6b). Thus, the concentration of APY0201 at this experimental dose could be consistent with the observed therapeutic effect in the IL-10−/− cell transfer colitis model.
In summary, a chemical proteomics approach using a highly sensitive DNLC–MS/MS system and bait compounds equipped with the FLAG epitope was used in this investigation, and further study demonstrated that the associated protein PIKfyve kinase was the possible target protein of APY0201, which is a selective and potent IL-12/23 production inhibitor. The data by researchers at Novartis supported these discussions. Characterization showed that APY0201 was a potent, highly selective, ATP-competitive PIKfyve kinase inhibitor that strongly inhibited IL-12/23 production in vitro and ameliorated inflammation in an experimental model of colitis. The structure–activity relationship study on APY0201, its pharmacological profile in vitro and in vivo, the strategy for target identification, and the biological characterization of PIKfyve kinase as an anti-inflammatory drug target were presented. The results from this investigation should be useful in drug discovery targeted at novel agents for treating autoimmune and inflammatory diseases. Our findings provide a new understanding of the function of PIKfyve kinase in the IL-12/23 production pathway and IL-12/23–driven inflammatory disease pathologies. In addition, these findings support the development of a selective PIKfyve kinase inhibitor as a therapeutic modality for autoimmune disorders, such as IBD.[1]

C23H23N7O

413.48

413.196

C, 66.81; H, 5.61; N, 23.71; O, 3.87

1232221-74-7

56927660

Light yellow to yellow solid powder

3.52

79.94

1

7

5

31

593

0

O1C([H])([H])C([H])([H])N(C2=C([H])C(N([H])/N=C(\[H])/C3=C([H])C([H])=C([H])C(C([H])([H])[H])=C3[H])=NC3=C([H])C(C4C([H])=C([H])N=C([H])C=4[H])=NN23)C([H])([H])C1([H])[H]

RFZQYGBLRIKROZ-PCLIKHOPSA-N

InChI=1S/C23H23N7O/c1-17-3-2-4-18(13-17)16-25-27-21-15-23(29-9-11-31-12-10-29)30-22(26-21)14-20(28-30)19-5-7-24-8-6-19/h2-8,13-16H,9-12H2,1H3,(H,26,27)/b25-16+

(E)-4-(5-(2-(3-methylbenzylidene)hydrazinyl)-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-7-yl)morpholine

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)

配方 1 中的溶解度: ≥ 2 mg/mL (4.84 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2 mg/mL (4.84 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2 mg/mL (4.84 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。