BT424

Hematopoietic cell kinase (HCK)

与其他SFKs相比，BT424在EC50为12 uM时对HCK有更强的选择性抑制（图7B、C、D）。研究人员对BT424进行了细胞毒性研究，发现在50 uM时对RAW264.7巨噬细胞和足细胞没有任何细胞毒性（图7E、F）。研究人员对BT424进行了稳定性测试，将100 mM的DMSO稀释至25 uM，并在室温下保存24小时，HPLC检测未发现降解（小于3%）。这些数据表明，研究人员开发了HCK更多选择的抑制剂BT424，可能是靶向化合物。[1]

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HCK抑制剂BT424降低巨噬细胞M1极化、增殖和迁移 [1]
首先，研究人员测试了BT424对bmdm自噬的影响。BT424不溶于水，研究人员将其放入100mm的DMSO中，在培养基中稀释至25um处理细胞。研究人员发现，BT424处理可以提高自噬活性，提高LC3II/LC3I比率，降低P62水平（图8A）。通过iNOS和CD206的M1和M2标记物的western blot显示，BT424抑制HCK激活可降低BMDM中巨噬细胞M1促炎极化，增加M2极化（图8B）。接下来，研究人员测量了BT424对巨噬细胞增殖的影响。研究人员首先在Raw264.7（图8C）细胞中进行了MTT实验，发现BT424显著降低了这些细胞的增殖。研究人员还在Raw264.7中使用碘化丙啶（PI）来测量细胞周期，发现BT424处理增加了G0/G1期，减少了G2/M期（图8D）。在Raw264.7中进行Click-iT™EdU细胞增殖试验（图8E）和Ki67 IF染色（图8F）。与对照细胞相比，BT424处理后EdU和Ki67阳性细胞减少。研究人员对Raw264.7细胞进行划痕实验，发现BT424显著降低了这些细胞的迁移能力（图8G）。这些数据表明BT424通过抑制HCK活性可以抑制巨噬细胞M1促炎极化、增殖和迁移。

HCK特异性抑制剂BT424改善UUO模型炎症和肾纤维化[1]
研究人员首先以25 mg/kg的BT424每天灌胃WT小鼠，连续1个月测试了HCK抑制剂的毒性。根据小鼠的体重变化、行为和身体活动，他们没有观察到任何明显的毒性。此外，研究人员通过IDEXX生物分析公司（Westbrook, ME 04092）的常规化学小组测试，对载药组和bt424处理小鼠的血清进行检测，未发现肝酶、肾功能、脂质谱和血糖水平有明显变化（补充表S1）。这些数据表明，BT424治疗不会对这些小鼠产生明显的毒性。研究人员随后测试了BT424在uuo诱导的纤维化中的作用。小鼠术前1天至术后7天每天灌胃BT424，剂量为25 mg/kg或载药（n = 5）。在H&E染色中，我们发现BT424处理减少了UUO小鼠肾小管的形态学损伤（图9A）。Masson和Col1a1 - if染色证实BT424治疗减轻了UUO肾脏的肾纤维化（图9A， B）。BT424治疗小鼠UUO肾脏中Col1a1、纤维连接蛋白和FSP-1 mRNA表达显著降低（图9C）。UUO处理BT424小鼠，f4 /80阳性巨噬细胞数量显著减少（图9D）。Western blot显示，由于巨噬细胞总数减少和M1极化减少，iNOS减少（图9E）。在BT424处理的肾脏中，由于巨噬细胞总数的减少，CD206减少，但在巨噬细胞数量调整后，由于BT424调节巨噬细胞，CD206增加，表明从M1向M2极化倾斜（图9E）。通过LC3和F4/80共染色以及巨噬细胞LC3的定量，BT424处理小鼠UUO肾脏巨噬细胞的自噬活性增加（图9F）。这些数据证实了BT424在UUO模型中通过减少巨噬细胞数量减轻纤维化，并通过自噬减少巨噬细胞炎症M1极化。

自噬测量[1]
按照生产商的方案，用PloyJet将自噬LC3 HiBiT报告基因转染到HEK293细胞中。然后用Nano-Glo HiBiT荧光试剂检测LC3报告基因。荧光信号随负载蛋白量的变化而变化。对于Raw264.7和BMDM，自噬诱导剂PP242 (1 uM)，自噬小体降解抑制剂巴菲霉素A1 (baafa1) (100 nM)，自噬抑制剂3-甲基腺嘌呤（3MA） （5 mM）处理24 h，用WB测量LC3和P62。在自噬刺激前2小时，加入HCK抑制剂BT424和达沙替尼预处理细胞。

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EGFR与整合素信号通路研究[1]
WT和HCK小鼠的BMDMs在第5天用1% FBS DMEM和L929饥饿过夜。然后用100 ng/ml的EGF处理细胞15和30分钟，然后用PBS冷洗冰收获。然后用细胞裂解液进行western blot检测EGFR信号相关蛋白。为了整合素信号传导，将BMDMs在胶原预包被的10厘米培养皿中接种1小时。IF染色和western blot检测F-actin和pY20与SYK。

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趋化因子阵列[1]
使用Proteome Profiler小鼠趋化因子阵列试剂盒，按照制造商的说明，使用WT和HCK KO小鼠BMDM培养基进行化学阵列。7天的BMDM用100 ng/ml LPS和50 ng/ml IFNγ极化24 h， WT BMDM在M1极化前用HCK抑制剂达沙替尼预处理2 h。

MTT和碘化丙啶对细胞增殖的影响[1]
培养4 d的BMDMs进行MTT和PI流动试验。使用碘化丙啶染色液和MTT，并遵循制造商的协议。采用tune流式细胞仪和FlowJo v10.8.0进行流式数据分析。BT424处理Raw 264.7 24 h后进行细胞增殖试验。

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二维和三维迁移试验[1]
化验。实验前一天，将RAW 264.7细胞和BMDMs涂于6个孔板上，饥饿过夜。用含0.5% FBS的饥饿培养基抑制细胞增殖。划痕是用200毫升吸管头划的。用0.5 FBS的饥饿培养基代替全培养基，减少细胞增殖。分别于划痕后0 h、1 h、3 h、6 h、12 h、24 h拍照。用ImageJ包“Wound_healing_size_tool”测量未愈合区域的距离。

Animal studies [1]
HCK exon3 loxp flanked KO mouse were developed at EuMMCR in Germany (HCK ES Cell Clone: HEPD0510). These mice were crossed with tissue specific Cre mice (CMV-cre B6.C-Tg (CMV-cre)1Cgn/J006054; LysM-cre B6.129P2-Lyz2tm1(cre)Ifo/J) to generate HCK KO specifically in tubular cells and macrophages. C57BL/6 J WT mice also from Jackson Laboratory. All mice were maintained in our animal facility at Mount Sinai under controlled environmental conditions: 12/12 light/dark cycle, ambient temperature 20–25 °C.
UUO model in WT, HCK KO, and HCK inhibitor mice was performed following our previous paper14. BT424 was made in 250 mg/ml in DMSO as stock, then was diluted to 2.5 mg/ml with PBS before gavage mice for final concentration of 25 mg/kg body weight. Unilateral IRI with contralateral nephrectomy (uIRIx) model was performed following the papers69,70, briefly, artery and vein of right kidney was tied, and the right kidney was removed. Then the left kidney’s artery and vein were clamped for 25 min and released. At the end of mice models, the mice were IP injected with 100 mg/kg ketamine and 10 mg/kg xylazine and then perfused with cold PBS for tissue collection.

[1]. HCK induces macrophage activation to promote renal inflammation and fibrosis via suppression of autophagy. Nat Commun. 2023 Jul 18;14(1):4297.

Renal inflammation and fibrosis are the common pathways leading to progressive chronic kidney disease (CKD). We previously identified hematopoietic cell kinase (HCK) as upregulated in human chronic allograft injury promoting kidney fibrosis; however, the cellular source and molecular mechanisms are unclear. Here, using immunostaining and single cell sequencing data, we show that HCK expression is highly enriched in pro-inflammatory macrophages in diseased kidneys. HCK-knockout (KO) or HCK-inhibitor decreases macrophage M1-like pro-inflammatory polarization, proliferation, and migration in RAW264.7 cells and bone marrow-derived macrophages (BMDM). We identify an interaction between HCK and ATG2A and CBL, two autophagy-related proteins, inhibiting autophagy flux in macrophages. In vivo, both global or myeloid cell specific HCK-KO attenuates renal inflammation and fibrosis with reduces macrophage numbers, pro-inflammatory polarization and migration into unilateral ureteral obstruction (UUO) kidneys and unilateral ischemia reperfusion injury (IRI) models. Finally, we developed a selective boron containing HCK inhibitor which can reduce macrophage pro-inflammatory activity, proliferation, and migration in vitro, and attenuate kidney fibrosis in the UUO mice. The current study elucidates mechanisms downstream of HCK regulating macrophage activation and polarization via autophagy in CKD and identifies that selective HCK inhibitors could be potentially developed as a new therapy for renal fibrosis.[1]

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In summary, we unravel a mechanism connecting the SFK HCK to progressive kidney IF/TA in CKD and CAI, by regulating autophagy within macrophages, altering their polarization, proliferation, and migration into diseased kidney in response to injury. We also developed a non-toxic specific HCK inhibitor ie a target-to-hit compound BT424, and demonstrated its regulation of macrophage function leading to attenuation of progressive renal fibrosis.[1]

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Our studies suggest that among the members of SFK, HCK expresses is highly regulated in the macrophages of diseased kidney. On other hand, FYN expresses mostly in podocytes and regulates podocyte function by phosphorylation of nephrin. We have previously shown that dasatinib induces proteinuria in lupus nephritis mice likely through inhibition of FYN-induced podocyte injury. Therefore, it is critical for us to develop more selective inhibitors of HCK, which will not affect FYN activity. We described here BT424, a relatively selective inhibitor of HCK and BT424 does not affect podocyte injury and therefore, we believe that BT424 is a better drug to target HCK as an anti-inflammatory and anti-fibrosis therapy in patients with kidney disease.
Our study has several limitations. We demonstrated that HCK is the main SFK in macrophages and highly regulated in kidney disease, but we could not rule out the role of other SFK members in kidney disease. We are also aware that macrophages have multiple functions in kidney disease. Recently, macrophages to myofibroblasts trans-differentiation have also been described and we will test whether this process is also regulated by HCK in our future studies. Also, total F4/80-positive cells, which includes macrophages (infiltrated and resident) but also some dendritic cells, were significantly reduced in the HCK KO mice with UUO. It would be specifically interesting to study the crosstalk between macrophages, tubular cells, and fibroblasts in the context of HCK knockout in future work. LysM-cre is not macrophage specific because the LysM promoter also expresses in neutrophils. Previous studies suggest that HCK regulates neutrophil activation and migration. Therefore, future studies are required to distinguish the roles of HCK in macrophages from neutrophils in kidney inflammation and fibrosis. We used HCK inhibitor BT424 to treat the mice before the injury, indicating the prevention of kidney injury or fibrosis in these mouse models. Future studies are also required to determine whether treatment of the mice after disease onset can reverse the kidney injury and fibrosis.
In summary, we demonstrate a critical role of HCK in regulation of macrophage function in the context of kidney inflammation and fibrosis. Using global- and cell-specific- HCK-KO models and by developing a selective boron containing HCK inhibitor, we demonstrate the therapeutic potential of this pathway in progressive kidney disease.[1]

C22H15BCL2N2O2

421.08

420.0603

2755180-37-9

168510578

White to off-white solid powder

42.8Ų

1

3

3

29

649

0

B1(N=C(NO1)C2=CC3=C(C(=CC(=C3)Cl)Cl)OC2C4=CC=CC=C4)C5=CC=CC=C5

SYIZHZBXDOQNIR-UHFFFAOYSA-N

InChI=1S/C22H15BCl2N2O2/c24-17-11-15-12-18(22-26-23(29-27-22)16-9-5-2-6-10-16)20(14-7-3-1-4-8-14)28-21(15)19(25)13-17/h1-13,20H,(H,26,27)

4-(6,8-dichloro-2-phenyl-2H-chromen-3-yl)-2-phenyl-5H-1,3,5,2-oxadiazaborole

BT424; BT-424; 2755180-37-9;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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；
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