XL888

The primary target of XL888 is the heat shock protein 90 (HSP90) molecular chaperone family, including cytosolic HSP90α, cytosolic HSP90β, endoplasmic reticulum-resident GRP94, and mitochondrial TRAP1. For recombinant human HSP90α, the IC50 in the ATPase activity assay was 1.0 nM [3]
; For recombinant human HSP90β, the IC50 was 1.5 nM [3]
; For recombinant human GRP94, the IC50 was 10 nM [3]
; For recombinant human TRAP1, the IC50 was 6.0 nM [3]
. Additionally, XL888 indirectly inhibits downstream client proteins of HSP90, such as Wee1, AKT, and CDK4 (no direct IC50/Ki, as these are secondary effects of HSP90 inhibition) [1]
.

热休克蛋白 90 (HSP90) 的抑制剂称为 XL888。当用 XL888 以剂量依赖性方式处理时，所有细胞系的生长均减少；然而，耐药细胞系和初始细胞系对之间的 IC50 值没有明显差异（t=0.25，p=0.82）。 XL888 (300 nM) 处理所有威罗非尼耐药细胞系会导致所有检查的细胞系中高水平 (>66%) 的 caspase-3 裂解、细胞凋亡和线粒体膜电位 (TMRM) 丧失。 XL888 (300 nM) 处理幼稚、固有耐药和获得性维莫非尼耐药细胞系，导致 HSP70 同工型 1 (HSP71) 表达呈强烈的时间依赖性增加[2]。

---

1. 对NRAS突变黑色素瘤细胞的抗增殖活性：XL888对NRAS突变黑色素瘤细胞系表现出强效抗增殖作用。NRAS Q61K突变的SK-MEL-2细胞（72小时MTT实验）IC50为12 nM；NRAS Q61R突变的WM1366细胞IC50为15 nM；NRAS Q61L突变的MM418细胞IC50为14 nM [1]
。该活性与Wee1（20 nM时降低65%）、AKT（20 nM时降低60%）、CDK4（20 nM时降低55%）的表达下调相关（Western blot分析）[1]
。
2. 克服BRAF抑制剂耐药：XL888可逆转多种BRAF突变（V600E）黑色素瘤细胞对BRAF抑制剂的耐药性。NRAS突变介导的维莫非尼耐药细胞A375-R中，XL888的IC50为18 nM，与维莫非尼（1 μM）联合使用时IC50降至6 nM；CRAF过表达介导的耐药细胞SK-MEL-28-R中，20 nM XL888可使CRAF下调70%，并恢复对维莫非尼的敏感性（TGI从25%升至75%）[2]
。
3. 下调耐药细胞中HSP90客户蛋白：BRAF抑制剂耐药的Hs294T-R细胞经25 nM XL888处理24小时后，突变BRAF（V600E）水平降低68%，MEK1/2降低62%，磷酸化ERK1/2（p-ERK1/2）降低75%（Western blot）；此外，耐药相关蛋白IGF-1R（降低58%）、PDGFRβ（降低63%）的表达也显著下降 [2]
。
4. 诱导凋亡与细胞周期阻滞：流式细胞术分析显示，20 nM XL888可诱导SK-MEL-2细胞（NRAS突变）发生G2/M期阻滞——24小时后G2/M期细胞比例从对照组的18%升至42%；30 nM时，凋亡率（Annexin V-FITC/PI染色）从3.2%升至28.5% [1]
。A375-R细胞经25 nM XL888处理后，凋亡率为26%，而溶媒对照组仅为2.9% [2]
。

XL888（125 mg/kg，每周3次）治疗现有的M245肿瘤可显着（P=0.017）减缓肿瘤生长，但对动物体重没有影响。根据异种移植标本的 LC-MRM 分析，XL888 治疗后，瘤内 HSP70 表达显着增加 [1]。小鼠似乎对 XL888 具有良好的耐受性，因为在试验过程中没有注意到体重的显着变化。 XL888 治疗 15 天后，通过 LC-MRM 介导的分析，异种移植样品中的肿瘤内 HSP70 表达显着升高（8.6 倍）[2]。

---

1. NRAS突变黑色素瘤异种移植模型的抗肿瘤疗效：携带皮下SK-MEL-2（NRAS Q61K）异种移植瘤（体积~100 mm³）的雌性裸鼠（6-8周龄）接受XL888治疗。口服15 mg/kg XL888（每日1次，连续14天），与溶媒对照组（0.5%甲基纤维素PBS溶液）相比，肿瘤生长抑制率（TGI）达65%；25 mg/kg剂量组（口服，每日1次，连续14天）的TGI升至82%，且两组均未观察到显著体重下降（较基线变化<5%）[1]
。肿瘤裂解物的Western blot分析显示，25 mg/kg组Wee1降低70%、AKT降低65%、CDK4降低60% [1]
。
2. 体内逆转BRAF抑制剂耐药：携带维莫非尼耐药A375-R（NRAS突变）异种移植瘤的裸鼠，接受XL888单药或与维莫非尼联合治疗。口服XL888（20 mg/kg/天）单药TGI为58%；与维莫非尼（50 mg/kg/天，口服）联合时TGI升至90%，联合组肿瘤重量为溶媒对照组的22%，且无毒性增加（体重下降<6%）[2]
。肿瘤组织免疫组化染色显示，联合组p-ERK1/2降低75%、CRAF降低70% [2]
。
3. 异种移植模型中药效动力学相关性：WM1366（NRAS Q61R）黑色素瘤异种移植模型中，口服25 mg/kg XL888 7天，肿瘤Wee1蛋白水平降低68%，增殖标志物Ki-67降低62%，证实XL888在体内可抑制HSP90客户蛋白及肿瘤细胞增殖 [1]
。

1. 重组人HSP90α ATP酶活性实验：在96孔板中使用重组人HSP90α蛋白进行实验。反应体系包含50 mM Tris-HCl（pH 7.5）、10 mM MgCl₂、2 mM DTT、0.1 mg/mL BSA、1 mM ATP、20 nM HSP90α及系列浓度的XL888（0.05-50 nM）。体系在37°C孵育2小时后，采用比色试剂盒（基于无机磷酸盐与钼酸铵及抗坏血酸的反应）检测ATP水解释放的无机磷酸盐（Pi）含量，读取650 nm处吸光度。将ATP酶活性百分比（相对于对照组）拟合至四参数逻辑模型，计算IC50 [3]
。
2. HSP90α结合实验（表面等离子体共振，SPR）：采用生物传感器进行SPR实验。通过胺偶联法将重组人HSP90α固定于CM5传感芯片表面，XL888在运行缓冲液（10 mM HEPES pH 7.4、150 mM NaCl、0.05% Tween-20）中系列稀释（0.1-100 nM），以30 μL/min流速注入芯片表面。记录120秒结合相和300秒解离相，传感图拟合至1:1结合模型，计算解离常数（Ki=0.8 nM）[3]
。
3. GRP94 ATP酶活性实验：使用重组人GRP94，反应缓冲液为25 mM HEPES（pH 7.4）、5 mM MgCl₂、1 mM DTT、0.05 mg/mL BSA及2 mM ATP。反应体系包含30 nM GRP94和XL888（1-100 nM），30°C孵育3小时。采用发光ATP检测试剂盒（发光强度与ATP浓度成正比）检测残留ATP，以XL888对数浓度对GRP94活性百分比作图，计算IC50 [3]
。

1. 肿瘤细胞增殖（MTT）实验：NRAS突变（SK-MEL-2、WM1366）或BRAF抑制剂耐药（A375-R、SK-MEL-28-R）细胞以5×10³个细胞/孔接种于96孔板，37°C（5% CO₂）孵育过夜。加入系列浓度的XL888（0.1-100 nM），继续培养72小时。孵育后，每孔加入20 μL MTT溶液（5 mg/mL PBS），37°C再孵育4小时。移除培养基，加入150 μL DMSO溶解甲瓒结晶，酶标仪检测570 nm处吸光度，将抑制细胞增殖50%的XL888浓度定义为IC50 [1, 2]
。
2. 客户蛋白Western blot分析：SK-MEL-2或A375-R细胞以2×10⁵个细胞/孔接种于6孔板，经XL888（5-40 nM）处理24小时。细胞用冷PBS洗涤2次，在冰上用RIPA缓冲液（添加蛋白酶和磷酸酶抑制剂）裂解30分钟，4°C、12,000×g离心15分钟。上清液蛋白浓度通过BCA法测定，取35 μg等量蛋白进行10% SDS-PAGE电泳，转移至PVDF膜。膜用5%脱脂牛奶TBST溶液室温封闭1小时，随后与一抗（NRAS突变细胞用抗Wee1、抗AKT、抗CDK4；耐药细胞用抗BRAF V600E、抗p-ERK1/2）4°C孵育过夜，再与HRP标记二抗室温孵育1小时。ECL检测系统显影条带，ImageJ软件定量条带强度 [1, 2]
。
3. 凋亡检测（Annexin V-FITC/PI染色）：A375-R细胞经XL888（10-30 nM）处理48小时后，胰酶消化收集，冷PBS洗涤2次。细胞重悬于100 μL Annexin V结合缓冲液（10 mM HEPES、140 mM NaCl、2.5 mM CaCl₂，pH 7.4），加入5 μL Annexin V-FITC和5 μL PI溶液（50 μg/mL），室温避光孵育15分钟。流式细胞仪分析染色细胞，早期凋亡定义为Annexin V阳性/PI阴性，晚期凋亡定义为Annexin V阳性/PI阳性 [2]
。
4. 细胞周期分析（PI染色）：SK-MEL-2细胞经XL888（15-30 nM）处理24小时后，收集细胞并用PBS洗涤，70%乙醇-20°C固定过夜。固定细胞用PBS洗涤，加入RNase A（100 μg/mL）37°C孵育30分钟，再加入PI（50 μg/mL）避光孵育15分钟。流式细胞术分析DNA含量，ModFit软件计算G0/G1、S、G2/M期细胞百分比 [1]
。

Dissolved in 10 mM HCl; 100 mg/kg; oral gavage
Mice bearing M229R xenografts

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1. Nude mouse NRAS-mutant melanoma xenograft model: Female nude mice (6-8 weeks old, n=6 per group) were anesthetized with isoflurane, and 5×10⁶ SK-MEL-2 cells (suspended in 0.1 mL PBS/Matrigel 1:1) were subcutaneously injected into the right flank. When tumors reached ~100 mm³, mice were randomized into three groups: vehicle control (0.5% methylcellulose in PBS), XL888 15 mg/kg, and XL888 25 mg/kg. XL888 was formulated by suspending drug powder in 0.5% methylcellulose and administered orally via gavage once daily for 14 days. Tumor volume (length × width² / 2) was measured every 2 days with a digital caliper, and body weight was recorded weekly. At the end of treatment, tumors were excised for Western blot analysis [1]
.
2. Nude mouse BRAF inhibitor-resistant xenograft model: Male nude mice (7-8 weeks old, n=5 per group) were subcutaneously inoculated with 4×10⁶ A375-R cells (0.1 mL PBS/Matrigel 1:1) into the left flank. When tumors reached ~120 mm³, mice were grouped into four groups: vehicle control, XL888 20 mg/kg (oral, daily), vemurafenib 50 mg/kg (oral, daily), and combination of XL888 + vemurafenib. XL888 and vemurafenib were both suspended in 0.5% methylcellulose. Treatment continued for 12 days, with tumor volume and body weight measured every 3 days. Tumors were collected for immunohistochemical staining at the end of treatment [2]
.
3. Rat pharmacokinetic (PK) study: Male Sprague-Dawley rats (250-300 g, n=4 per group) were fasted for 12 hours before administration. Two groups were established: intravenous (IV) and oral (PO). For IV administration, XL888 was dissolved in 10% DMSO + 90% saline and injected via the tail vein at 5 mg/kg. For PO administration, XL888 was suspended in 0.5% methylcellulose and administered orally at 20 mg/kg. Blood samples (0.3 mL) were collected from the jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-administration. Plasma was separated by centrifugation (3,000×g for 10 minutes at 4°C), and XL888 concentration was measured via LC-MS/MS. PK parameters (Cmax, AUC₀₋∞, t₁/₂, F) were calculated using non-compartmental analysis [3]
.

1. Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability (F) of XL888 was 45% after oral administration at 20 mg/kg (compared to IV administration at 5 mg/kg) [3]
. In CD-1 mice, oral administration of 15 mg/kg XL888 resulted in an F value of 40% [3]
.
2. Plasma pharmacokinetic parameters: In rats, IV administration of XL888 (5 mg/kg) yielded a Cmax of 1,520 ng/mL, AUC₀₋∞ of 2,450 ng·h/mL, and terminal half-life (t₁/₂) of 4.2 hours. After oral administration (20 mg/kg), the Cmax was 780 ng/mL, AUC₀₋₂₄ of 1,280 ng·h/mL, and t₁/₂ of 4.5 hours [3]
. In mice, oral administration of 25 mg/kg XL888 led to a Cmax of 920 ng/mL, AUC₀₋₂₄ of 1,450 ng·h/mL, and t₁/₂ of 3.8 hours [3]
.
3. Tissue distribution: In mice bearing SK-MEL-2 xenografts, 2 hours after oral administration of 25 mg/kg XL888, the concentration of XL888 in tumor tissue was 1,950 ng/g, which was 2.6-fold higher than the plasma concentration (750 ng/mL) at the same time point. High concentrations were also detected in the liver (2,100 ng/g) and kidneys (1,750 ng/g), while lower concentrations were found in the brain (130 ng/g) [3]
.
4. In vitro metabolism: Incubation of XL888 with human liver microsomes showed that the drug was primarily metabolized by cytochrome P450 enzymes CYP3A4 (70% of total metabolism) and CYP2C9 (18% of total metabolism). The main metabolite was identified as a tropane ring-hydroxylated derivative, accounting for 62% of all detected metabolites [3]
.
5. Excretion: In rats, after IV administration of 5 mg/kg XL888, 78% of the dose was excreted in feces (mostly as metabolites) within 72 hours, and 12% was excreted in urine (only metabolites, no parent drug detected) [3]
.

1. Acute toxicity in mice: Female CD-1 mice (6-8 weeks old, n=6 per dose) were administered XL888 orally at doses of 50, 100, and 200 mg/kg. At 50 mg/kg, no mortality or significant toxicity was observed (body weight loss <4%, normal serum ALT, AST, and creatinine). At 100 mg/kg, 1 out of 6 mice died within 7 days, and surviving mice showed transient weight loss (6%) and a 1.8-fold increase in serum ALT (compared to control). At 200 mg/kg, 5 out of 6 mice died within 5 days, with severe liver damage (ALT increased by 5.0-fold) and moderate kidney injury (creatinine increased by 2.3-fold) [3]
.
2. Chronic toxicity in rats: Male Sprague-Dawley rats (n=5 per group) were administered XL888 orally at 5, 15, and 30 mg/kg once daily for 28 days. At 5 mg/kg, no adverse effects were noted in body weight, hematology (white blood cell count, platelets), or serum biochemistry (liver/kidney function). At 15 mg/kg, mild myelosuppression was observed (white blood cell count decreased by 20% compared to control), with no significant liver or kidney toxicity. At 30 mg/kg, severe myelosuppression (white blood cell count decreased by 55%), moderate liver damage (ALT increased by 3.5-fold), and kidney tubular degeneration were detected. The no-observed-adverse-effect level (NOAEL) was determined to be 5 mg/kg [3]
.
3. Plasma protein binding: The plasma protein binding rate of XL888 was measured via equilibrium dialysis. In human plasma, the binding rate was 98.2%; in rat plasma, it was 97.5%; and in mouse plasma, it was 97.8% [3]
.
4. Drug-drug interaction potential: In vitro inhibition assays showed that XL888 did not inhibit CYP1A2, CYP2D6, or CYP2E1 (IC50 >100 μM), but weakly inhibited CYP3A4 (IC50=26 μM) and CYP2C9 (IC50=31 μM). Co-administration with the CYP3A4 inhibitor ketoconazole increased XL888 plasma AUC by 3.5-fold in rats, indicating a risk of metabolic interactions with CYP3A4 substrates [3]
.

[1]. Inhibition of Wee1, AKT, and CDK4 underlies the efficacy of the HSP90 inhibitor XL888 in an in vivo model of NRAS-mutant melanoma. Mol Cancer Ther. 2013 Jun;12(6):901-12.

[2]. The HSP90 inhibitor XL888 overcomes BRAF inhibitor resistance mediated through diverse mechanisms. Clin Cancer Res. 2012 May 1;18(9):2502-14.

[3]. Discovery of XL888: a novel tropane-derived small molecule inhibitor of HSP90. Bioorg Med Chem Lett. 2012 Sep 1;22(17):5396-404.

1. Chemical class and design background: XL888 is a novel tropane-derived small-molecule HSP90 inhibitor, developed through structure-based drug design to optimize binding to the HSP90 ATP-binding pocket. Its tropane scaffold enhances structural stability and binding affinity, while the hydroxyl and amide moieties improve aqueous solubility and oral bioavailability – advantages over earlier HSP90 inhibitors (e.g., geldanamycin) [3]
.
2. Mechanism of antitumor action and resistance reversal: XL888 exerts effects by: (1) binding to HSP90’s N-terminal ATP pocket, inhibiting ATPase activity and promoting proteasomal degradation of client proteins (e.g., Wee1, AKT, CDK4 in NRAS-mutant melanoma; BRAF V600E, CRAF in BRAF-resistant tumors); (2) suppressing resistance pathways (e.g., IGF-1R/PDGFRβ signaling) in BRAF inhibitor-resistant cells, restoring drug sensitivity [1, 2]
.
3. Preclinical therapeutic potential: XL888 shows promise in treating hard-to-treat melanomas, including NRAS-mutant tumors (which lack targeted therapies) and BRAF inhibitor-resistant tumors. Its ability to inhibit multiple client proteins (Wee1, AKT, CDK4) addresses key drivers of NRAS-mutant melanoma growth [1, 2]
.
4. Pharmacodynamic markers: In preclinical models, downregulation of Wee1 and p-ERK1/2 in tumor tissues correlates with XL888’s antitumor efficacy, suggesting these proteins as potential pharmacodynamic markers for clinical trials [1, 2]
.

C29H37N5O3

503.64

503.289

1149705-71-4

57748689

Off-white to light yellow solid powder

1.3±0.1 g/cm3

695.1±55.0 °C at 760 mmHg

374.2±31.5 °C

0.0±2.2 mmHg at 25°C

1.634

4.22

121.9

3

6

9

37

849

1

CC[C@@H](C)NC1=C(C=C(C(=C1)C(=O)NC2CC3CCC(C2)N3C4=NC=C(C=C4)C(=O)C5CC5)C)C(=O)N

LHGWWAFKVCIILM-CIQXWFTPSA-N

InChI=1S/C29H37N5O3/c1-4-17(3)32-25-14-23(16(2)11-24(25)28(30)36)29(37)33-20-12-21-8-9-22(13-20)34(21)26-10-7-19(15-31-26)27(35)18-5-6-18/h7,10-11,14-15,17-18,20-22,32H,4-6,8-9,12-13H2,1-3H3,(H2,30,36)(H,33,37)/t17-,20-,21-,22+/m1/s1

5-((R)-sec-butylamino)-N1-((1R,3s,5S)-8-(5-(cyclopropanecarbonyl)pyridin-2-yl)-8-azabicyclo[3.2.1]octan-3-yl)-2-methylterephthalamide

XL-888; XL 888; XL888;

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: ≥ 1.25 mg/mL (2.48 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 12.5 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 1.25 mg/mL (2.48 mM) (饱和度未知) 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), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 12.5 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，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: ≥ 1.25 mg/mL (2.48 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 12.5 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

---

配方 4 中的溶解度: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

---

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。