Geldanamycin   中文名称: 格尔德霉素

Hsp90 (Kd = 1.2 μM); anticancer antimicrobial/antibiotic
The primary target of Geldanamycin is the heat shock protein 90 (HSP90) molecular chaperone family, with high affinity for the N-terminal ATP-binding pocket of cytosolic HSP90α/β. For recombinant human HSP90α, the dissociation constant (Ki) measured by isothermal titration calorimetry (ITC) was 1.2 nM [4]
; The binding affinity (KD) determined by surface plasmon resonance (SPR) was 1.5 nM [4]
. No significant binding to other chaperones (e.g., HSP70, HSP60) was observed [4]
. In LNCaP prostate cancer cells and primary cortical neurons, Geldanamycin exerted biological effects by inhibiting HSP90 [2, 3]
.

格尔德霉素大大延迟并减少了 RAW264.7 细胞中 Viperin 的诱导，表明 IRF3 在此过程中发挥作用 [1]。在培养的原代神经元中，苯醌安沙霉素格尔德霉素可防止氧糖剥夺 (OGD)/zVAD 疗法引起的神经元损伤。更重要的是，格尔德霉素以依赖时间和浓度的方式降低 RIP1 蛋白水平。此外，格尔德霉素会降低 Hsp90 蛋白水平，从而导致 RIP1 蛋白不稳定。因此，格尔德霉素治疗后，RIP1 蛋白水平下降，但 RIP1 mRNA 水平保持不变 [2]。在天然物质中发现的第一个已知的 Hsp90 抑制剂是格尔德霉素。它通过与其N端ATP酶结构域结合，抑制Hsp90的分子伴侣功能，并通过凋亡机制，极大地导致肿瘤细胞死亡[3]。

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1. 对氧糖剥夺（OGD）诱导的神经元损伤的保护作用：原代大鼠皮质神经元经OGD（1% O₂、无糖培养基）处理4小时并联合caspase抑制剂zVAD（20 μM）后，Geldanamycin（50、200 nM）呈剂量依赖性减轻神经元损伤。200 nM剂量下，细胞膜损伤标志物乳酸脱氢酶（LDH）释放量较OGD+zVAD组降低45%；Western blot分析显示，200 nM Geldanamycin可下调RIP1（降低58%）和切割型caspase-8（降低62%），同时上调抗凋亡蛋白Bcl-2（升高2.3倍）[2]
。
2. 对前列腺癌细胞的抗增殖活性：Geldanamycin抑制人前列腺癌LNCaP细胞增殖，72小时MTT实验测得IC50为2.5 μM [3]
。Western blot分析显示，Geldanamycin（1-5 μM）呈剂量依赖性下调HSP90客户蛋白：3 μM剂量下，雄激素受体（AR）水平较溶媒对照组降低65%，Akt降低60%，CDK4降低55%；同时，HSP90抑制诱导的应激反应蛋白HSP70表达升高3.1倍 [3]
。
3. 抑制H5N1流感病毒复制：人肺泡上皮A549细胞感染H5N1病毒（感染复数MOI=0.1）后，Geldanamycin（0.5-2 μM）可减少病毒复制。1 μM剂量下，感染48小时后病毒载量（qPCR检测病毒M基因）降低70%；ELISA结果显示，1 μM Geldanamycin还可减少促炎细胞因子TNF-α（降低55%）和IL-6（降低60%）的分泌 [5]
。
4. 与HSP90的结合特性：等温滴定量热法（ITC）显示，Geldanamycin与重组人HSP90α的结合化学计量比为1:1，结合焓变（ΔH）为-28 kcal/mol [4]
；表面等离子体共振（SPR）证实，Geldanamycin特异性结合HSP90的ATP结合口袋，与HSP70无显著结合（KD>100 μM）[4]
。

在携带 FRE/erbB-2 肿瘤的小鼠中，格尔德霉素 (50 mg/kg) 对 p185 相关磷酸酪氨酸水平显示出 30% 的抑制作用。

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1. 对H5N1诱导的小鼠急性呼吸窘迫综合征（ARDS）的保护作用：6-8周龄雌性BALB/c小鼠经鼻感染H5N1病毒（100 PFU/只），感染后12小时开始，通过腹腔注射给予Geldanamycin（1 mg/kg、5 mg/kg），每日1次，连续5天。5 mg/kg组小鼠感染14天后的生存率为80%，而溶媒对照组（含5% DMSO的0.9%生理盐水）生存率仅为20% [5]
。肺组织病理分析显示，5 mg/kg Geldanamycin可减少65%的肺泡出血和炎症细胞浸润，感染7天后肺组织病毒载量（qPCR）降低75% [5]
。此外，与对照组相比，5 mg/kg Geldanamycin可使血清TNF-α降低60%，IL-6降低65% [5]
。

通过测定培养基中乳酸脱氢酶（LDH）水平来评估皮层神经元的存活率。在各种处理后，收集培养基并将其滴在VITROS Chemistry Products LDH DT载玻片上，用自动生化免疫分析仪测量LDH水平[2]。

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1. 等温滴定量热法（ITC）检测HSP90结合：实验在25°C下使用微量热仪进行。样品池（1.4 mL）含10 μM重组人HSP90α（溶于20 mM Tris-HCl pH 7.5、150 mM NaCl、2 mM DTT缓冲液）；注射器（250 μL）含100 μM Geldanamycin（同缓冲液溶解）。将Geldanamycin分25次、每次10 μL注入样品池，两次注射间隔2分钟以实现信号平衡。记录每次注射的热变化（μcal/sec），数据拟合至单位点结合模型，计算解离常数（Ki）和结合化学计量比 [4]
。
2. 表面等离子体共振（SPR）检测HSP90结合：通过胺偶联法将重组人HSP90α共价固定于CM5传感芯片（目标固定水平约500共振单位RU）。运行缓冲液为10 mM HEPES pH 7.4、150 mM NaCl、0.05% Tween-20、2 mM DTT。Geldanamycin在运行缓冲液中系列稀释（0.1-50 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。传感图通过减去空白参考池（无HSP90固定）信号校正，拟合至1:1朗缪尔结合模型，确定平衡解离常数（KD）[4]
。

蛋白质印迹分析[2]
从细胞中提取蛋白质后，通过双辛可宁酸蛋白质测定法测定蛋白质浓度。然后等量的蛋白质（100 μg）在10%聚丙烯酰胺凝胶电泳上分离并电转移到硝化纤维膜上。用5%脱脂干乳封闭印迹，并与抗RIP1、Hsp90和β-肌动蛋白的一级抗体在4°C下孵育过夜。洗涤后，将印迹与第二抗体孵育1 h.用奥德赛系统开发印迹。用软件Image J（V1.40）进行谱带的密度分析

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免疫沉淀[2]
对采集的神经元细胞进行裂解和超声处理。蛋白质浓度通过二辛可宁酸蛋白质法测定。将等量的蛋白质用于免疫沉淀。将样品与蛋白A琼脂糖珠孵育2小时 h，然后稍微离心。将上清液与RIP1抗体在4°C下振荡孵育过夜。第二天，加入蛋白A琼脂糖珠并振荡2 h，温度为4°C。离心2小时后 10分钟 000 g、 用裂解缓冲液洗涤珠子三次。最后，将裂解缓冲液和4×样品缓冲液加入珠粒中，并在96°C下加热5 min。对收集的上清液进行蛋白质印迹分析。

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1. 原代皮质神经元培养与OGD损伤模型：从E18 Sprague-Dawley大鼠胚胎分离皮质神经元，接种于多聚-L-赖氨酸包被的96孔板（5×10⁴个细胞/孔）和6孔板（2×10⁵个细胞/孔），用添加B27的神经基础培养基培养7天。OGD处理时，培养基更换为无糖Earle平衡盐溶液（EBSS），细胞置于低氧培养箱（1% O₂、5% CO₂、94% N₂）中孵育4小时。Geldanamycin（50-200 nM）在OGD前1小时加入，并在OGD期间维持。OGD后更换为正常神经基础培养基，继续培养24小时。通过比色法（490 nm）检测LDH释放评估细胞损伤；Western blot检测凋亡相关蛋白（RIP1、切割型caspase-8、Bcl-2）[2]
。
2. LNCaP细胞增殖与Western blot实验：人前列腺癌LNCaP细胞接种于96孔板（5×10³个细胞/孔，用于增殖实验）和6孔板（2×10⁵个细胞/孔，用于蛋白分析），用含10% FBS的RPMI 1640培养基培养。加入Geldanamycin（0.5-10 μM），培养72小时。增殖实验中，每孔加入20 μL MTT溶液（5 mg/mL PBS），孵育4小时后加入DMSO溶解甲瓒结晶，570 nm处测吸光度计算IC50。蛋白分析中，细胞用含蛋白酶抑制剂的RIPA缓冲液裂解，35 μg蛋白经10% SDS-PAGE分离后转移至PVDF膜，用抗AR、Akt、CDK4和HSP70抗体孵育，ECL显影 [3]
。
3. H5N1感染A549细胞实验：A549细胞接种于24孔板（1×10⁵个细胞/孔），用含10% FBS的DMEM培养至80%融合，感染H5N1病毒（MOI=0.1）1小时。去除未结合病毒，加入含Geldanamycin（0.5-2 μM）的培养基。感染24和48小时后，收集细胞上清液，通过qPCR检测H5N1 M基因测定病毒载量，ELISA检测促炎细胞因子（TNF-α、IL-6）；细胞裂解后Western blot检测病毒NP蛋白 [5]
。

Dissolved in DMSO; 50 mg/kg; i.p. injection
FRE/erbB-2 tumors in nu/nu mice

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1. H5N1 influenza virus-infected mouse model: Female BALB/c mice (6-8 weeks old, n=10 per group) were anesthetized with isoflurane and intranasally inoculated with 100 PFU of H5N1 virus in 50 μL of PBS. Twelve hours post-infection, mice were randomly divided into three groups: vehicle control (0.9% saline with 5% DMSO), Geldanamycin 1 mg/kg, and Geldanamycin 5 mg/kg. Geldanamycin was dissolved in DMSO first, then diluted with 0.9% saline to a final DMSO concentration of 5%, and administered via intraperitoneal injection once daily for 5 consecutive days. Mice were monitored daily for survival and body weight changes. At 7 days post-infection, 3 mice per group were euthanized, and lung tissues were collected for histopathological analysis (H&E staining), viral load measurement (qPCR), and Western blot (viral NP protein). Serum was collected to detect inflammatory cytokines (TNF-α, IL-6) by ELISA [5]
.

1. Acute toxicity in H5N1-infected mice: In the H5N1 mouse model, Geldanamycin was administered at 1 mg/kg and 5 mg/kg via intraperitoneal injection for 5 days. No significant body weight loss (<5% change from baseline) or mortality related to Geldanamycin was observed. Serum levels of alanine transaminase (ALT) and creatinine were within normal ranges, indicating no obvious liver or kidney toxicity [5]
.

[1]. Viperin inhibits rabies virus replication via reduced cholesterol and sphingomyelin and is regulated upstream by TLR4. Sci Rep. 2016 Jul 26;6:30529.

[2]. RIP1 mediates the protection of Geldanamycin on neuronal injury induced by oxygen-glucosedeprivation combined with zVAD in primary cortical neurons. J Neurochem. 2012 Jan;120(1):70-7.

[3]. 17-ABAG, a novel Geldanamycin derivative, inhibits LNCaP-cell proliferation through heat shock protein 90 inhibition. Int J Mol Med. 2015 Aug;36(2):424-32.

[4]. Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin. J Med Chem. 1999 Jan 28;42(2):260-6.

[5]. Geldanamycin Reduces Acute Respiratory Distress Syndrome and Promotes the Survival of Mice Infected with the Highly Virulent H5N1 Influenza Virus. Front Cell Infect Microbiol. 2017 Jun 15;7:267.

Geldanamycin is an ansamycin consisting of a 19-membered macrocyle incorporating a benzoquinone ring and a lactam functionality. It shows antimicrobial activity against many Gram-positive and some Gram-negative bacteria. It has a role as an antiviral agent, an antineoplastic agent, an antimicrobial agent, a cysteine protease inhibitor and a Hsp90 inhibitor. It is an ansamycin, a carbamate ester, an organic heterobicyclic compound and a member of 1,4-benzoquinones.
Geldanamycin has been reported in Humicola fuscoatra and Streptomyces hygroscopicus with data available.
Geldanamycin is a benzoquinone antineoplastic antibiotic isolated from the bacterium Streptomyces hygroscopicus. Geldanamycin binds to and inhibits the cytosolic chaperone functions of heat shock protein 90 (HSP90). HSP90 maintains the stability and functional shape of many oncogenic signaling proteins; the inhibition of HSP90 promotes the proteasomal degradation of oncogenic signaling proteins that may be over-expressed or overactive in tumor cells. (NCI04)

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1. Chemical class and structural basis: Geldanamycin is a natural ansamycin-type antibiotic derived from Streptomyces hygroscopicus. Its structure contains a benzoquinone ring and a macrolide chain, which enables specific binding to the N-terminal ATP-binding pocket of HSP90—this binding induces conformational changes in HSP90, disrupting its chaperone function and promoting degradation of client proteins [4]
.
2. Mechanism of biological effects: Geldanamycin exerts its effects by: (1) inhibiting HSP90 ATPase activity, leading to proteasomal degradation of client proteins (e.g., AR, Akt in cancer cells; viral proteins in H5N1-infected cells); (2) reducing inflammatory responses (downregulating TNF-α, IL-6) in H5N1-induced ARDS [3, 5]
; (3) protecting neurons by inhibiting RIP1-mediated apoptotic pathways in OGD-induced neuronal injury [2]
.
3. Therapeutic potential: Geldanamycin shows preclinical potential in: (1) treating hormone-dependent cancers (e.g., prostate cancer) via inhibiting HSP90 client proteins (AR, Akt) [3]
; (2) alleviating viral-induced ARDS (e.g., H5N1 influenza) by reducing viral replication and inflammation [5]
; (3) neuroprotection in ischemic brain injury models (e.g., OGD-induced neuronal damage) [2]
.

C29H40N2O9

560.64

560.273

C, 62.13; H, 7.19; N, 5.00; O, 25.68

30562-34-6

5288382

Light yellow to orange solid

1.2±0.1 g/cm3

783.9±60.0 °C at 760 mmHg

255 °C

427.9±32.9 °C

0.0±6.2 mmHg at 25°C

1.559

2

163.48

3

9

5

40

1150

6

COC(C(C=C1NC(/C(C)=C/C=C/[C@H](OC)[C@@H](OC(N)=O)/C(C)=C/[C@H](C)[C@H]2O)=O)=O)=C(C[C@H](C[C@@H]2OC)C)C1=O

QTQAWLPCGQOSGP-KSRBKZBZSA-N

InChI=1S/C29H40N2O9/c1-15-11-19-25(34)20(14-21(32)27(19)39-7)31-28(35)16(2)9-8-10-22(37-5)26(40-29(30)36)18(4)13-17(3)24(33)23(12-15)38-6/h8-10,13-15,17,22-24,26,33H,11-12H2,1-7H3,(H2,30,36)(H,31,35)/b10-8-,16-9+,18-13+/t15-,17+,22+,23+,24-,26+/m1/s1

(4E,6Z,8S,9S,10E,12S,13R,14S,16R)-13-hydroxy-8,14,19-trimethoxy-4,10,12,16-tetramethyl-3,20,22-trioxo-2-azabicyclo[16.3.1]docosa-1(21),4,6,10,18-pentaen-9-yl carbamate

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: 2.5 mg/mL (4.46 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 25.0 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。

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配方 2 中的溶解度: 2.5 mg/mL (4.46 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 25.0 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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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。