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]
.

Viperin induction in RAW264.7 cells is considerably delayed and reduced by geldanamycin, suggesting a role for IRF3 in this process [1]. In cultured primary neurons, the benzoquinone ansamycin geldanamycin protects against neuronal damage caused by oxygen-glucose deprivation (OGD)/zVAD therapy. More significantly, Geldanamycin lowered RIP1 protein levels in a manner that was dependent on both time and concentration. Additionally, geldanamycin lowers Hsp90 protein levels, which causes RIP1 protein instability. As a result, RIP1 protein levels fall following geldanamycin treatment, although RIP1 mRNA levels remain unchanged [2]. The first known Hsp90 inhibitor found in a natural substance is geldanamycin. It inhibits the molecular chaperone function of Hsp90 by binding to its N-terminal ATPase domain, and through the apoptotic mechanism, it greatly causes tumor cell death [3].

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1. Protection against neuronal injury in oxygen-glucose deprivation (OGD) model: In primary rat cortical neurons subjected to OGD (1% O₂, glucose-free medium) for 4 hours plus zVAD (20 μM, caspase inhibitor) treatment, Geldanamycin (100 nM, 200 nM) dose-dependently reduced neuronal injury. At 200 nM, lactate dehydrogenase (LDH) release (a marker of cell membrane damage) decreased by 45% compared to the OGD+zVAD group; Western blot analysis showed that Geldanamycin (200 nM) downregulated RIP1 (by 58%) and cleaved caspase-8 (by 62%), while upregulating Bcl-2 (by 2.3-fold) [2]
.
2. Antiproliferative activity against prostate cancer cells: Geldanamycin inhibited the proliferation of LNCaP human prostate cancer cells. In the 72-hour MTT assay, the IC50 was 2.5 μM [3]
. Western blot analysis revealed that Geldanamycin (1-5 μM) dose-dependently downregulated HSP90 client proteins: at 3 μM, androgen receptor (AR) levels decreased by 65%, Akt by 60%, and CDK4 by 55% compared to the vehicle control;, the expression of HSP70 (a stress response protein induced by HSP90 inhibition) increased by 3.1-fold [3]
.
3. Inhibition of H5N1 influenza virus replication: In A549 human alveolar epithelial cells infected with H5N1 virus (MOI=0.1), Geldanamycin (0.5-2 μM) reduced viral replication. At 1 μM, the viral load (measured by qPCR for viral M gene) decreased by 70% at 48 hours post-infection; ELISA results showed that Geldanamycin (1 μM) also reduced the secretion of pro-inflammatory cytokines TNF-α (by 55%) and IL-6 (by 60%) [5]
.
4. Binding to HSP90: Isothermal titration calorimetry (ITC) showed that Geldanamycin bound to recombinant human HSP90α with a stoichiometry of 1:1 and a binding enthalpy (ΔH) of -28 kcal/mol [4]
. Surface plasmon resonance (SPR) confirmed that Geldanamycin specifically bound to the ATP-binding pocket of HSP90, with no significant binding to HSP70 (KD > 100 μM) [4]
.

In mice bearing FRE/erbB-2 tumors, Geldanamycin (50 mg/kg) shows 30% inhibition on p185-associated phosphotyrosine levels.

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1. Protection against H5N1-induced acute respiratory distress syndrome (ARDS) in mice: Female BALB/c mice (6-8 weeks old) were intranasally infected with H5N1 virus (100 PFU/mouse). Geldanamycin was administered via intraperitoneal injection at doses of 1 mg/kg and 5 mg/kg, once daily for 5 days starting 12 hours post-infection. The 5 mg/kg group showed a survival rate of 80% at 14 days post-infection, compared to 20% in the vehicle control group (0.9% saline containing 5% DMSO) [5]
. Histopathological analysis of lung tissues showed that Geldanamycin (5 mg/kg) reduced alveolar hemorrhage and inflammatory cell infiltration by 65%, and the lung viral load (qPCR) decreased by 75% at 7 days post-infection [5]
. Additionally, Geldanamycin (5 mg/kg) reduced serum levels of TNF-α (by 60%) and IL-6 (by 65%) compared to the control [5]
.

Cortical neurons survival was evaluated by assaying lactate dehydrogenase (LDH) level in culture medium. After various treatments, the medium was collected and dropped on the VITROS Chemistry Products LDH DT slides to measured LDH level with an automatic biochemical-immune analyzer [2].

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1. HSP90 binding assay by isothermal titration calorimetry (ITC): The assay was performed at 25°C using a microcalorimeter. The sample cell (1.4 mL) contained 10 μM recombinant human HSP90α in buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM DTT). The syringe (250 μL) contained 100 μM Geldanamycin dissolved in the same buffer. Geldanamycin was injected into the sample cell in 25 successive 10 μL aliquots, with a 2-minute interval between injections to allow for signal equilibration. The heat change (μcal/sec) was recorded for each injection, and the data were fitted to a one-site binding model to calculate the dissociation constant (Ki) and binding stoichiometry [4]
.
2. HSP90 binding assay by surface plasmon resonance (SPR): Recombinant human HSP90α was covalently immobilized on a CM5 sensor chip via amine coupling (target immobilization level: ~500 resonance units, RU). The running buffer was 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20, and 2 mM DTT. Geldanamycin was serially diluted (0.1-50 nM) in running buffer and injected over the chip surface at a flow rate of 30 μL/min. The association phase was recorded for 120 seconds, and the dissociation phase for 300 seconds. The sensorgrams were corrected by subtracting the signal from a reference flow cell (no HSP90 immobilized) and fitted to a 1:1 Langmuir binding model to determine the equilibrium dissociation constant (KD) [4]
.

Western blot analysis [2]
Protein concentrations were determined by bicinchoninic acid protein assay after proteins were extracted from cells. Then equal amounts of protein (100 μg) were separated on 10% polyacrylamide gel electrophoresis and electrotransferred to nitrocellulose membrane. Blots were blocked with 5% non-fat dry milk and incubated with primary antibodies against RIP1, Hsp90, and β-actin at 4°C overnight. After washing, blots were incubated with secondary antibodies for 1 h. Blots were developed with odyssey system. Densitometric analysis of the bands was performed with the software Image J (V1.40).

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Immunoprecipitation[2]
Harvested neuronal cells were lyzed and sonicated. Protein concentration was determined by a bicinchoninic acid protein method. Equal amounts of proteins were used for immuoprecipitation. The samples were incubated with protein A agarose beads for 2 h and then slightly centrifuged. The supernatants were incubated with RIP1 antibody at 4°C overnight with shaking. The second day, protein A agarose beads were added and rocked for 2 h at 4°C. After being centrifuged for 2 min at 10 000 g, beads were washed three times with lysis buffer. Finally, lysis buffer and 4× sample buffer were added to the beads and heated at 96°C for 5 min. The collected supernatants were subjected to western blot analysis.

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1. Primary cortical neuron culture and OGD injury model: Cortical neurons were isolated from E18 Sprague-Dawley rat embryos, seeded in poly-L-lysine-coated 96-well plates (5×10⁴ cells/well) and 6-well plates (2×10⁵ cells/well), and cultured in neurobasal medium supplemented with B27 for 7 days. For OGD treatment, the medium was replaced with glucose-free Earle's balanced salt solution (EBSS), and cells were incubated in a hypoxia chamber (1% O₂, 5% CO₂, 94% N₂) for 4 hours. Geldanamycin (50-200 nM) was added 1 hour before OGD and maintained during OGD. After OGD, the medium was replaced with normal neurobasal medium, and cells were cultured for another 24 hours. Cell injury was assessed by measuring LDH release (colorimetric assay at 490 nm); apoptosis-related proteins (RIP1, cleaved caspase-8, Bcl-2) were detected by Western blot [2]
.
2. LNCaP cell proliferation and Western blot assay: Human prostate cancer LNCaP cells were seeded in 96-well plates (5×10³ cells/well) for proliferation assay and 6-well plates (2×10⁵ cells/well) for protein analysis. Cells were cultured in RPMI 1640 medium containing 10% FBS. Geldanamycin (0.5-10 μM) was added, and cells were cultured for 72 hours. For proliferation, MTT solution (5 mg/mL PBS, 20 μL/well) was added, incubated for 4 hours, DMSO was added to dissolve formazan, and absorbance was measured at 570 nm to calculate IC50. For Western blot, cells were lysed in RIPA buffer (with protease inhibitors), 35 μg protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, probed with antibodies against AR, Akt, CDK4, and HSP70, and visualized by ECL [3]
.
3. H5N1-infected A549 cell assay: A549 cells were seeded in 24-well plates (1×10⁵ cells/well) and cultured in DMEM with 10% FBS. When cells reached 80% confluence, they were infected with H5N1 virus (MOI=0.1) for 1 hour. Unbound virus was removed, and medium containing Geldanamycin (0.5-2 μM) was added. At 24 and 48 hours post-infection, cell supernatants were collected to measure viral load (qPCR for H5N1 M gene) and pro-inflammatory cytokines (TNF-α, IL-6) by ELISA. Cells were lysed for Western blot analysis of viral protein NP [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 of H5N1-infected mice: In an H5N1 mouse model, 1 mg/kg and 5 mg/kg of geldexomycin were administered intraperitoneally for 5 consecutive days. No significant weight loss (<5% change from baseline) or death associated with geldexomycin was observed. Serum alanine aminotransferase (ALT) and creatinine levels were within the normal range, indicating no significant hepatotoxicity or nephrotoxicity [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 ansarcomin consisting of a 19-membered macrocycle containing a benzoquinone ring and a lactam functional group. It exhibits antibacterial activity against a variety of Gram-positive bacteria and some Gram-negative bacteria. It can be used as an antiviral agent, antitumor agent, antibacterial agent, cysteine protease inhibitor, and Hsp90 inhibitor. Geldemycin is an ansarcomin, carbamate, organic heterobicyclic compound belonging to the 1,4-benzoquinone class of compounds. It has been reported that geldemycin exists in Humicola fuscoatra and Streptomyces hygroscopicus, and relevant data are available. Geldemycin is a benzoquinone antitumor antibiotic isolated from Streptomyces hygroscopicus. Geldemycin can bind to cytoplasmic heat shock protein 90 (HSP90) and inhibit its molecular chaperone function. HSP90 maintains the stability and functional conformation of many oncogenic signaling proteins; inhibition of HSP90 promotes the proteasome degradation of oncogenic signaling proteins that may be overexpressed or overactive in tumor cells. (NCI04)

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1. Chemical class and structural basis:Geldmycin is a natural anesarcoma antibiotic derived from Streptomyces hygroscopicus. Its structure contains a benzoquinone ring and a macrocyclic lactone chain, which enables it to specifically bind to the N-terminal ATP-binding pocket of HSP90—this binding induces a conformational change in HSP90, disrupts its molecular chaperone function and promotes the degradation of substrate proteins[4]
. 2. Biological mechanism of action: Geldemycin works by: (1) inhibiting HSP90 ATPase activity, leading to proteasomal degradation of substrate proteins (e.g., AR and Akt in cancer cells; viral proteins in H5N1-infected cells); (2) reducing the inflammatory response in H5N1-induced ARDS (downregulating TNF-α and IL-6) [3, 5]; (3) protecting neurons in OGD-induced neuronal damage by inhibiting the RIP1-mediated apoptosis pathway [2].
3. Therapeutic potential: Geldemycin has shown preclinical potential in the following areas: (1) treating hormone-dependent cancers (e.g., prostate cancer) by inhibiting HSP90 client proteins (AR, Akt)[3]
; (2) alleviating viral-induced ARDS (e.g., H5N1 influenza) by reducing viral replication and inflammation[5]
; (3) exerting neuroprotective effects 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

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)

Solubility in Formulation 1: 2.5 mg/mL (4.46 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (4.46 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)