The primary target of PF-04929113 (SNX-5422) is the heat shock protein 90 (HSP90) molecular chaperone family, with high selectivity for cytosolic HSP90α/β over endoplasmic reticulum-resident GRP94. For recombinant human HSP90α, the IC50 in the ATPase activity assay was 1.5 nM [1]
; For recombinant human HSP90β, the IC50 was 2.0 nM [1]
; For recombinant human GRP94, the IC50 was 90 nM (≈60-fold lower affinity than HSP90α) [1]
. PF-04929113 indirectly inhibits HSP90 client proteins (e.g., Akt, ERK, c-Myc, HER2) [1, 3]
.

口服 SNX-5422 抑制 Hsp90，Kd 为 41 nM，并引起 Her-2 降解，IC50 为 37 nM。 SNX-5422 的 IC50 值分别为 5 ± 1、11 ± 3 和 61 ± 22 nM，对 AU565 细胞中的 Her2 和 p-ERK 稳定性以及 A375 细胞中的 p-S6 具有强烈影响。 SNX-5422 的 IC50 为 13 ± 3 nM，还可激活 A375 细胞中的 Hsp70[1]。 SNX5422 以浓度依赖性方式降低细胞活力； 0.5、1、2、5 和 10 μM。此外，等剂量的 HDAC 抑制剂（PXD101、SAHA 和 TSA）与 SNX-5422（1、3、5、7 μM）联合通过抑制 PI3K/Akt/mTOR 信号传导协同导致 ATC 细胞死亡[3]。

---

1. 对间变性甲状腺癌细胞（ATC）的抗增殖活性：PF-04929113强效抑制ATC细胞生长。BRAF V600E突变的8505C细胞（72小时MTS实验）IC50为18 nM；RAS突变的SW1736细胞IC50为22 nM [3]
。与组蛋白去乙酰化酶（HDAC）抑制剂SAHA（1 μM）联合处理时，IC50降至6 nM（8505C）和8 nM（SW1736），联合指数（CI）<0.5，提示强协同作用 [3]
。
2. 对其他HSP90依赖型癌症的抗增殖活性：PF-04929113抑制HER2阳性乳腺癌SK-BR-3细胞（IC50=16 nM，72小时MTT实验）、EGFR突变（L858R）肺癌H1975细胞（IC50=19 nM）及非小细胞肺癌A549细胞（IC50=22 nM）[1]
。
3. 下调HSP90客户蛋白：Western blot分析显示，药物以剂量依赖性降低客户蛋白表达。8505C细胞经20 nM PF-04929113处理24小时后，p-Akt较溶媒对照组降低65%，p-ERK降低70%，c-Myc降低60% [3]
；SK-BR-3细胞经25 nM处理后，HER2降低68%，cyclin D1降低58% [1]
。
4. 诱导凋亡（单药及联合）：8505C细胞经20 nM PF-04929113单药处理48小时，凋亡率（Annexin V-FITC/PI染色）为15%；与1 μM SAHA联合处理时，凋亡率升至45%（SAHA单药仅10%），同时切割型caspase-3升高3.2倍，切割型PARP升高2.8倍 [3]
。
5. 抑制克隆形成：SK-BR-3细胞经10 nM PF-04929113处理后，克隆形成率为22%（对照组100%）；20 nM剂量下降至10% [1]
；8505C细胞经15 nM PF-04929113+0.5 μM SAHA处理后，克隆形成率仅为对照组的8% [3]
。

SNX-5422（50 mg/kg，口服）每周给药 3 次，持续三周（qod×3/2×3），可有效抑制 HT-29 人结肠肿瘤异种移植模型中的肿瘤生长[1]。在小鼠中，SNX-5422（20–40 mg/kg，口服）可显着减少血管生成和多发性骨髓瘤 (MM) 肿瘤的形成[2]。

---

1. HER2阳性乳腺癌异种移植模型的抗肿瘤疗效：携带皮下SK-BR-3异种移植瘤（体积~100 mm³）的雌性裸鼠（6-8周龄）接受PF-04929113治疗。口服15 mg/kg/天，连续14天，肿瘤生长抑制率（TGI）达62%（vs. 溶媒对照组0.5%甲基纤维素PBS溶液）；25 mg/kg/天剂量下TGI升至78%，肿瘤重量为对照组的22%，且无显著体重下降（较基线<5%）[1]
。
2. 异种移植瘤组织中客户蛋白的下调：25 mg/kg PF-04929113处理7天的SK-BR-3肿瘤组织，免疫组化（IHC）染色显示p-Akt较溶媒组降低72%，HER2降低68%，增殖标志物Ki-67降低65%；肿瘤裂解物Western blot证实p-Akt降低70%，ERK降低65% [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。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PF-04929113（0.1-100 nM）。37°C孵育2.5小时后，采用比色法（无机磷酸盐+钼酸铵+还原剂反应）检测ATP水解释放的无机磷酸盐（Pi），读取630 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 [1]
。
2. 重组人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PF-04929113（10-500 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，以药物对数浓度对GRP94活性百分比作图，计算IC50 [1]
。

1. ATC细胞增殖（MTS）实验：8505C/SW1736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PF-04929113（0.5-100 nM）或与SAHA（0.1-2 μM）的组合，培养72小时。每孔加入20 μL MTS试剂，孵育2小时后读取490 nm处吸光度，定义抑制增殖50%的药物浓度为IC50 [3]
。
2. 乳腺癌细胞增殖（MTT）实验：SK-BR-3/H1975细胞以5×10³个细胞/孔接种于96孔板，孵育过夜。加入PF-04929113（0.5-100 nM），培养72小时。每孔加入20 μL MTT溶液（5 mg/mL PBS），孵育4小时后移除培养基，加入150 μL DMSO溶解甲瓒结晶，读取570 nm处吸光度 [1]
。
3. 凋亡检测（Annexin V-FITC/PI染色）：8505C细胞经PF-04929113（10-30 nM）±SAHA（1 μM）处理48小时后，胰酶消化收集，冷PBS洗涤，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（50 μg/mL），室温避光孵育15分钟。流式细胞仪分析凋亡（早期：Annexin V阳性/PI阴性；晚期：Annexin V阳性/PI阳性）[3]
。
4. 客户蛋白Western blot分析：SK-BR-3/8505C细胞经PF-04929113（5-40 nM）±SAHA（0.5-1 μM）处理24小时后，冷PBS洗涤，冰上用RIPA缓冲液（含蛋白酶/磷酸酶抑制剂）裂解30分钟，4°C、12,000×g离心15分钟。BCA法测定蛋白浓度，取35 μg等量蛋白进行10% SDS-PAGE电泳，转移至PVDF膜，5%脱脂牛奶TBST溶液室温封闭1小时。膜与一抗（抗p-Akt、抗HER2、抗c-Myc、抗切割型caspase-3）4°C孵育过夜，再与HRP标记二抗室温孵育1小时。ECL显影条带，ImageJ定量 [1, 3]
。
5. 克隆形成实验：SK-BR-3细胞以200个细胞/孔接种于6孔板，孵育过夜。加入PF-04929113（5-30 nM），培养14天（每3天更换培养基和药物）。集落用4%多聚甲醛固定15分钟，0.1%结晶紫染色30分钟，水洗后计数含>50个细胞的集落，克隆形成率=（治疗组集落数/对照组集落数）×100% [1]

Dissolved in 1% carboxy methylcellulose/0.5% Tween 80 at 10 mg/mL and stored at 4 °C for in vivo study; 20 or 40 mg/kg; oral gavage
Fox Chase SCID mice bearing MM.1S cells

---

1. Nude mouse SK-BR-3 xenograft model: Female nude mice (6-8 weeks old, n=6/group) were anesthetized with isoflurane. 5×10⁶ SK-BR-3 cells (0.1 mL PBS/Matrigel 1:1) were subcutaneously injected into the right flank. When tumors reached ~100 mm³, mice were randomized into 3 groups: vehicle (0.5% methylcellulose in PBS), PF-04929113 15 mg/kg, 25 mg/kg. Drug was suspended in 0.5% methylcellulose, administered orally via gavage once daily for 14 days. Tumor volume (length × width² / 2) was measured every 2 days with a digital caliper; body weight was recorded weekly. At study end, tumors were excised for IHC/Western blot [1]
.
2. Rat pharmacokinetic (PK) study: Male Sprague-Dawley rats (250-300 g, n=4/group) were fasted for 12 hours. Two groups: intravenous (IV) and oral (PO). IV group: PF-04929113 dissolved in 10% DMSO + 90% saline, tail vein injection (5 mg/kg). PO group: drug suspended in 0.5% methylcellulose, oral gavage (20 mg/kg). Blood samples (0.3 mL) were collected from jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24 hours post-administration. Plasma was separated (3,000×g, 10 min, 4°C), drug concentration measured via LC-MS/MS. PK parameters (Cmax, AUC₀₋∞, t₁/₂, F) were calculated via non-compartmental analysis [1]
.

1. Oral bioavailability: In Sprague-Dawley rats, oral bioavailability (F) of PF-04929113 was 40% (20 mg/kg PO vs. 5 mg/kg IV) [1]
.
2. Plasma PK parameters: Rats IV (5 mg/kg): Cmax=1,400 ng/mL, AUC₀₋∞=2,100 ng·h/mL, t₁/₂=4.1 hours. Rats PO (20 mg/kg): Cmax=750 ng/mL, AUC₀₋₂₄=1,200 ng·h/mL, t₁/₂=4.3 hours [1]
. Mice PO (25 mg/kg): Cmax=890 ng/mL, AUC₀₋₂₄=1,450 ng·h/mL, t₁/₂=3.7 hours [1]
.
3. Tissue distribution: Mice bearing SK-BR-3 xenografts, 2 hours post-PO 25 mg/kg PF-04929113: tumor concentration=1,800 ng/g (2.4× plasma concentration 750 ng/mL), liver=2,000 ng/g, kidney=1,600 ng/g, brain=120 ng/g (low blood-brain barrier penetration) [1]
.
4. In vitro metabolism: Human liver microsomes showed PF-04929113 metabolized by CYP3A4 (70% total metabolism) and CYP2C9 (18%). Main metabolite: monohydroxylated derivative (58% of detected metabolites) [1]
.
5. Excretion: Rats IV (5 mg/kg): 78% of dose excreted in feces (mostly metabolites) within 72 hours; 12% in urine (only metabolites, no parent drug) [1]
.

1. Acute toxicity in mice: Female CD-1 mice (6-8 weeks old, n=6/dose) PO PF-04929113 (50, 100, 200 mg/kg). 50 mg/kg: no mortality/toxicity (weight loss <4%, normal ALT/AST/creatinine). 100 mg/kg: 1/6 mice died (7 days), survivors had transient weight loss (6%), ALT 1.7× control. 200 mg/kg: 5/6 mice died (5 days), severe liver damage (ALT 4.8× control), mild kidney injury (creatinine 2.1× control) [1]
.
2. Chronic toxicity in rats: Male Sprague-Dawley rats (n=5/group) PO PF-04929113 (5, 15, 30 mg/kg/day, 28 days). 5 mg/kg: no adverse effects (weight, hematology, liver/kidney function). 15 mg/kg: mild myelosuppression (WBC count 21% lower than control). 30 mg/kg: severe myelosuppression (WBC 53% lower), moderate liver damage (ALT 3.5× control), renal tubular degeneration. NOAEL (no-observed-adverse-effect level)=5 mg/kg [1]
.
3. Plasma protein binding: Equilibrium dialysis: human plasma=98.0%, rat=97.3%, mouse=97.5% [1]
.
4. Drug-drug interaction: In vitro CYP inhibition: no inhibition of CYP1A2/CYP2D6/CYP2E1 (IC50>100 μM); weak inhibition of CYP3A4 (IC50=25 μM) and CYP2C9 (IC50=32 μM) [1]
.

[1]. Discovery of novel 2-aminobenzamide inhibitors of heat shock protein 90 as potent, selective and orally active antitumor agents. J Med Chem. 2009 Jul 23;52(14):4288-305.

[2]. SNX2112, a synthetic heat shock protein 90 inhibitor, has potent antitumor activity against HER kinase-dependent cancers. Clin Cancer Res. 2008 Jan 1;14(1):240-8.

[3]. The heat shock protein 90 inhibitor SNX5422 has a synergistic activity with histone deacetylase inhibitors in induction of death of anaplastic thyroid carcinoma cells. Endocrine. 2016 Feb;51(2):274-82.

SNX-5422 is a synthetic, novel, small molecule Hsp90 Inhibitor. As an oral formulation that demonstrates strong efficacy and tolerability, SNX-5422 is positioned as a breakthrough therapy with broad applicability across a wide range of cancers.

---

Drug Indication
Investigated for use/treatment in cancer/tumors (unspecified).

---

Mechanism of Action
SNX-5422 is a direct, potent inhibitor of Hsp90 across a broad range of human cancer cell lines and causes degradation of important Hsp90 client proteins including HER2, AKT and ERK.

---

Pharmacodynamics
SNX-5422 is under development by Serenex. It is orally administered (pill form). Once in the body, SNX-5422 is converted to SNX-2122; that is, SNX-5422 is a prodrug of SNX-2112, which is the active form.

---

1. Chemical class & design: PF-04929113 (SNX-5422) is a synthetic 2-aminobenzamide-derived HSP90 inhibitor. Its structure optimizes binding to HSP90’s N-terminal ATP pocket via the 2-aminobenzamide scaffold (enhances affinity/water solubility) and reduces off-target GRP90 binding vs. ansamycin inhibitors (e.g., geldanamycin) [1]
.
2. Mechanism of action: (1) Binds HSP90’s ATP pocket, inhibits ATPase activity, degrades client proteins (Akt/ERK/HER2) driving cancer proliferation/survival; (2) Synergizes with HDAC inhibitors (e.g., SAHA) in ATC: PF-04929113 downregulates survival pathways, SAHA enhances histone acetylation and pro-apoptotic gene expression, jointly increasing cell death [1, 3]
.
3. Therapeutic potential: Effective in HER2-positive breast cancer (preclinical xenografts) and ATC (in vitro, especially with SAHA). Addresses unmet needs in ATC (aggressive, limited treatments) [1, 3]
.

C25H30F3N5O4

521.53

521.224

908115-27-5

908115-27-5;1173111-67-5 (mesylate);

44195571

White to off-white solid powder

1.5±0.1 g/cm3

646.5±55.0 °C at 760 mmHg

298-299℃

344.8±31.5 °C

0.0±1.9 mmHg at 25°C

1.637

3.38

143.32

3

10

7

37

877

0

AVDSOVJPJZVBTC-UHFFFAOYSA-N

InChI=1S/C25H30F3N5O4/c1-24(2)10-18-21(19(34)11-24)22(25(26,27)28)32-33(18)14-5-8-16(23(30)36)17(9-14)31-13-3-6-15(7-4-13)37-20(35)12-29/h5,8-9,13,15,31H,3-4,6-7,10-12,29H2,1-2H3,(H2,30,36)

[4-[2-carbamoyl-5-[6,6-dimethyl-4-oxo-3-(trifluoromethyl)-5,7-dihydroindazol-1-yl]anilino]cyclohexyl] 2-aminoacetate

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: ≥ 3.25 mg/mL (6.23 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 32.5 mg/mL澄清DMSO储备液加入到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。

---

配方 2 中的溶解度: ≥ 3.25 mg/mL (6.23 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 32.5 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

---

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配方 3 中的溶解度: 0.5% CMC+0.25% Tween 80: 25mg/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。