SLU-PP-1072

The target of SLU-PP-1072 is estrogen-related receptor α (ERRα) and estrogen-related receptor γ (ERRγ), and it acts as a selective inverse agonist for both targets. The dissociation constant (Ki) of SLU-PP-1072 with ERRα ligand-binding domain (LBD) is 14 nM, and with ERRγ LBD is 12 nM, as determined by fluorescence polarization assay[1]

1. 抗前列腺癌细胞增殖活性：在人前列腺癌细胞系（雄激素敏感性LNCaP细胞、雄激素非敏感性PC-3细胞）中，SLU-PP-1072可浓度依赖性抑制细胞增殖；采用MTT法检测72小时药物作用后的细胞存活率，结果显示其对LNCaP细胞的半数抑制浓度IC50为1.2 μM，对PC-3细胞的IC50为0.8 μM[1]
2. 诱导前列腺癌细胞凋亡：将LNCaP和PC-3细胞分别用1 μM、2 μM的SLU-PP-1072处理48小时后，通过Annexin V-FITC/PI双染色流式细胞术检测，发现凋亡细胞比例显著升高——PC-3细胞在2 μM药物处理时凋亡率较空白对照组升高约3.5倍，LNCaP细胞在2 μM药物处理时凋亡率较空白对照组升高约2.8倍；同时，Western blot检测显示，凋亡相关蛋白（cleaved caspase-3、cleaved PARP）的表达水平显著上调，抗凋亡蛋白Bcl-2的表达水平显著下调[1]
3. 抑制Warburg效应：SLU-PP-1072处理LNCaP和PC-3细胞后，可浓度依赖性降低细胞的葡萄糖摄取能力和乳酸生成量；以2-NBDG（荧光葡萄糖类似物）为探针检测葡萄糖摄取，2 μM药物处理时细胞葡萄糖摄取量较对照组降低40%-50%；通过乳酸检测试剂盒检测，2 μM药物处理时细胞乳酸生成量较对照组降低35%-45%；此外，Western blot和实时定量PCR（qPCR）检测显示，Warburg效应关键调控分子（葡萄糖转运体1 GLUT1、己糖激酶2 HK2、乳酸脱氢酶A LDHA）的蛋白及mRNA表达水平均显著下调[1]
4. 调控ERRα/γ下游靶基因表达：qPCR检测显示，SLU-PP-1072处理后，ERRα/γ下游与线粒体能量代谢相关的靶基因（细胞色素c氧化酶亚基4亚型1 COX4I1、ATP合成酶β亚基ATP5B）的mRNA表达水平显著降低；在PC-3细胞中，2 μM药物处理时COX4I1 mRNA表达量较对照组降低55%，ATP5B mRNA表达量较对照组降低48%[1]

采用6-8周龄雄性BALB/c裸鼠建立PC-3前列腺癌皮下移植瘤模型，当肿瘤体积达到约100 mm³时，将裸鼠随机分为3组（每组6只）：空白对照组（给予溶媒）、SLU-PP-1072低剂量组（25 mg/kg）、SLU-PP-1072高剂量组（50 mg/kg）；药物通过腹腔注射给药，每周给药5次，连续给药21天。实验结果显示：
1. SLU-PP-1072可剂量依赖性抑制移植瘤生长，50 mg/kg组给药结束时肿瘤体积较对照组减少62%，肿瘤重量较对照组减少58%[1]
2. 瘤组织免疫组化分析显示，50 mg/kg组瘤内细胞增殖标志物Ki-67的阳性表达率较对照组降低45%，凋亡标志物cleaved caspase-3的阳性表达率较对照组升高3倍[1]
3. 瘤组织匀浆检测显示，SLU-PP-1072处理组的葡萄糖摄取量、乳酸生成量较对照组显著降低，且GLUT1、HK2、LDHA的蛋白表达水平也显著下调[1]

采用荧光偏振（FP）实验检测SLU-PP-1072与ERRα/γ配体结合域（LBD）的结合亲和力，具体流程如下：
1. 制备重组人ERRα-LBD和ERRγ-LBD蛋白，确保蛋白纯度和活性符合实验要求[1]
2. 配置反应体系：将不同浓度的SLU-PP-1072（浓度范围0.1 nM-10 μM）与固定浓度的重组ERR-LBD蛋白（20 nM）、荧光标记的ERR激动剂配体（荧光素标记的GSK4716，10 nM）共同加入缓冲液（含50 mM Tris-HCl pH 7.5、100 mM NaCl、1 mM DTT、0.01% Tween-20）中，每组设3个复孔[1]
3. 反应体系在室温下孵育1小时，随后使用荧光偏振检测仪检测荧光偏振值（激发波长485 nm，发射波长535 nm）[1]
4. 以SLU-PP-1072浓度为横坐标，荧光偏振值为纵坐标绘制竞争结合曲线，采用GraphPad Prism软件通过非线性回归拟合曲线，计算药物与ERRα/γ-LBD的Ki值[1]

1. 细胞增殖实验（MTT法）：将LNCaP和PC-3细胞以5×10³个/孔的密度接种于96孔板，培养24小时后加入不同浓度的SLU-PP-1072（0.1 μM-10 μM），每组3个复孔；继续培养72小时后，每孔加入20 μL MTT溶液（5 mg/mL），37℃孵育4小时；弃去上清液，每孔加入150 μL DMSO溶解甲瓒结晶，酶标仪在490 nm波长处检测吸光度值；以药物浓度为横坐标，细胞存活率（相对于对照组）为纵坐标绘制量效曲线，计算IC50值[1]
2. 细胞凋亡实验（Annexin V-FITC/PI双染色流式细胞术）：将LNCaP和PC-3细胞以2×10⁵个/孔接种于6孔板，培养24小时后加入1 μM、2 μM的SLU-PP-1072，继续培养48小时；收集细胞，用预冷PBS洗涤2次，加入100 μL Binding Buffer重悬细胞，再加入5 μL Annexin V-FITC和5 μL PI染色液，室温避光孵育15分钟；加入400 μL Binding Buffer，流式细胞仪检测凋亡细胞比例[1]
3. 葡萄糖摄取实验：将细胞以5×10⁴个/孔接种于24孔板，药物处理24小时后弃去培养基，用无糖DMEM洗涤2次；加入含10 μM 2-NBDG的无糖DMEM，37℃孵育30分钟；PBS洗涤3次，0.25%胰酶消化细胞，PBS重悬后，流式细胞仪检测细胞内荧光强度（激发波长488 nm，发射波长530 nm），以荧光强度反映葡萄糖摄取能力[1]
4. 乳酸生成检测：细胞经药物处理48小时后，收集培养上清液；按照乳酸检测试剂盒说明书，将上清液与反应试剂混合，37℃孵育10分钟；酶标仪在570 nm波长处检测吸光度值，根据标准曲线计算乳酸浓度[1]
5. Western blot实验：细胞经药物处理后，用含蛋白酶抑制剂和磷酸酶抑制剂的RIPA裂解液提取总蛋白，BCA法测定蛋白浓度；取20-30 μg蛋白进行SDS-PAGE电泳，随后将蛋白转移至PVDF膜；5%脱脂牛奶封闭膜1小时，加入一抗（ERRα、GLUT1、HK2、LDHA、cleaved caspase-3、cleaved PARP、Bcl-2、β-actin）4℃孵育过夜；TBST洗涤3次后，加入荧光二抗室温孵育1小时；TBST洗涤3次，ECL化学发光试剂盒显影，ImageJ软件定量分析蛋白条带灰度值，以β-actin为内参计算目标蛋白相对表达水平[1]
6. qPCR实验：细胞经药物处理后，用Trizol试剂提取总RNA，逆转录合成cDNA；以cDNA为模板，使用SYBR Green荧光定量PCR试剂盒进行扩增（反应条件：95℃预变性5分钟，95℃变性15秒，60℃退火延伸30秒，共40个循环）；以GAPDH为内参基因，采用2^(-ΔΔCt)法计算目标基因（GLUT1、HK2、LDHA、COX4I1、ATP5B）的相对mRNA表达水平[1]

1. Establishment of xenograft model: Under sterile conditions, PC-3 cells in the logarithmic growth phase (1×10⁷ cells/0.2 mL) were subcutaneously injected into the right back of 6-8 week-old male BALB/c nude mice. After inoculation, the mental state, activity, and diet of the mice were observed daily. The long diameter (L) and short diameter (W) of the tumor were measured with a vernier caliper every week, and the tumor volume was calculated using the formula V = (L×W²)/2. Drug administration was initiated when the tumor volume reached approximately 100 mm³[1]
2. Drug preparation: The administration vehicle of SLU-PP-1072 was prepared by mixing DMSO, PEG400, and normal saline at a volume ratio of 1:4:5. The drug concentration was adjusted according to the administration dose (25 mg/kg, 50 mg/kg) and mouse weight (administration volume was calculated as 10 μL/g body weight) to ensure that the drug was completely dissolved and the solution was clear without precipitation[1]
3. Grouping and administration: Tumor-bearing nude mice were randomly divided into 3 groups (6 mice per group): control group (intraperitoneal injection of equal volume of vehicle), SLU-PP-1072 low-dose group (25 mg/kg, intraperitoneal injection), and SLU-PP-1072 high-dose group (50 mg/kg, intraperitoneal injection). The administration frequency was 5 times a week (Monday to Friday) for 21 consecutive days[1]
4. Sample collection and processing: After the end of administration, the body weight of the mice was measured, and then the mice were sacrificed by cervical dislocation. The tumor tissues were stripped and weighed. Part of the tumor tissues was fixed in 4% paraformaldehyde solution (for subsequent immunohistochemical analysis), and the other part was immediately frozen in liquid nitrogen and then transferred to a -80℃ refrigerator for storage (for Western blot, qPCR detection, and determination of glucose uptake and lactate production)[1]

During the PC-3 prostate cancer xenograft model experiment in nude mice (21 days), no obvious toxic reactions were observed in the SLU-PP-1072 treatment group (25 mg/kg, 50 mg/kg): 1. There was no significant difference in body weight between the groups before and after the experiment, and no weight loss was observed (which is usually considered a basic indicator of toxicity) [1] 2. The mice had normal daily activities and did not show abnormalities such as disheveled fur, lethargy, reduced appetite and water intake [1] 3. After sacrifice, the weight of the major organs (heart, liver, spleen, lungs, kidneys) of the mice was weighed, and there was no significant difference in organ weight between the groups. Plasma protein binding rate, liver and kidney function biochemical indicators (such as ALT, AST, BUN, Cr) or median lethal dose (LD50) were not detected in the literature [1]

[1]. A Selective ERRα/γ Inverse Agonist, SLU-PP-1072, Inhibits the Warburg Effect and Induces Apoptosis in Prostate Cancer Cells. ACS Chem Biol. 2020 Sep 18;15(9):2338-2345.

1. Mechanism of action: As a selective inverse agonist of ERRα/γ, SLU-PP-1072 binds to the ligand-binding domain of ERRα/γ, thereby inhibiting the transcriptional activity of ERRα/γ. This further downregulates the expression of two key downstream target genes: mitochondrial energy metabolism-related genes (COX4I1, ATP5B) and Warburg effect-related genes (GLUT1, HK2, LDHA). Ultimately, this leads to insufficient energy supply for prostate cancer cells and simultaneously activates the apoptosis pathway, thereby inhibiting tumor cell proliferation [1]. 2. Significance of research: ERRα/γ is highly expressed in prostate cancer (especially androgen-insensitive prostate cancer) and is associated with tumor progression and poor prognosis, making it a potential target for prostate cancer treatment. SLU-PP-1072 has been shown to have inhibitory activity against prostate cancer cells through in vitro and in vivo experiments, and can target ERRα/γ, providing a new candidate drug direction for the treatment of prostate cancer (especially refractory androgen-insensitive prostate cancer) [1]

336.06

C, 64.27; H, 3.60; N, 8.33; O, 14.27; S, 9.53

2285432-57-5

2285432-57-5

137518263

Light yellow to brown solid powder

2

5

3

24

456

0

DYMDZGMAYKMYCT-UHFFFAOYSA-N

InChI=1S/C18H12N2O3S/c21-13-4-1-11(2-5-13)15-7-8-16(23-15)18(22)20-12-3-6-14-17(9-12)24-10-19-14/h1-10,21H,(H,20,22)

N-(benzo[d]thiazol-6-yl)-5-(4-hydroxyphenyl)furan-2-carboxamide

SLUPP-1072; SLU-PP 1072; SLU PP-1072; SLU-PP1072; SLU-PP-1072

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

DMSO: ~125 mg/mL (~371.6 mM)

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；
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;
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