HSP90 (IC50 = 5 nM); Mitophagy; Autophagy
Heat shock protein 90 (HSP90) [5]
Androgen receptor (AR) [1]
Human epidermal growth factor receptor 2 [1][2]
Serine/threonine kinase 38 (STK38) [6]

Tanespimycin degrades HER2, Akt, and the G1 growth halt of prostate cancer cells that is dependent on retinoblastoma as well as mutant and wild-type AR. With IC50 values ranging from 25 to 45 nM (LNCaP, 25 nM; LAPC-4, 40 nM; DU-145, 45 nM; and PC-3, 25 nM), tantespimycin suppresses prostate cancer cell lines [1]. Tanespimycin (0.1–1 μM) causes breast cancer cells that overexpress ErbB2 to almost completely lose ErbB2[2]. Tanespimycin downregulates Bcl-2, Survivin, and Cyclin B1, and upregulates cleaved PARP. These effects prevent the development of CCA cells and cause G2/M cell cycle arrest and apoptosis[3].

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In prostate cancer cells (LNCaP, CWR22Rv1) overexpressing AR and HER-2, Tanespimycin (17-AAG) induced dose-dependent degradation of AR and HER-2 proteins (Western blot analysis), with >50% protein reduction at 1 μM after 24 hours [1]
The drug inhibited prostate cancer cell proliferation (MTT assay) with an IC50 of 0.2–0.5 μM and suppressed colony formation (colony assay) by ~70% at 0.3 μM [1]
In ErbB2-overexpressing breast cancer cells (SKBR3, BT474), Tanespimycin (17-AAG) enhanced ubiquitination of ErbB2 and promoted its degradation via the lysosomal pathway, leading to 80% reduction in ErbB2 protein at 1 μM (24 hours) [2]
It induced cytotoxicity in breast cancer cells with an IC50 of 0.3–0.7 μM (CCK-8 assay) and increased apoptotic rates to ~35% at 1 μM (Annexin V/PI staining) [2]
In human cholangiocarcinoma cells (QBC939, RBE), Tanespimycin (17-AAG) inhibited cell growth (IC50 = 0.4–0.6 μM, MTT assay) and induced apoptosis, as evidenced by caspase-3 activation and PARP cleavage (Western blot) [3]
The drug downregulated anti-apoptotic proteins (Bcl-2, survivin) and upregulated pro-apoptotic proteins (Bax) in cholangiocarcinoma cells [3]
In lymphoma cells (SU-DHL-4, OCI-Ly10) and lymphoma stem cells (LSC), Tanespimycin (17-AAG) selectively eradicated LSC with an IC50 of 0.15 μM, compared to IC50 = 0.5 μM for bulk lymphoma cells (flow cytometry-based viability assay) [4]
It depleted LSC by inducing proteasomal degradation of c-Myc and Notch1 (Western blot) and inhibiting self-renewal (sphere formation assay: ~60% reduction in sphere number at 0.2 μM) [4]
In human cholangiocarcinoma cells (HuCCT1), Tanespimycin (17-AAG) downregulated STK38 expression via inhibiting Sp1 transcription factor activity (EMSA and luciferase reporter assay) [6]
Combination with radiation enhanced radiosensitivity of cholangiocarcinoma cells, increasing apoptotic rates from 20% (radiation alone) to 45% (radiation + 0.3 μM 17-AAG) [6]

In prostate cancer xenografts, tantespimycin (25–200 mg/kg, ip) reduces AR, HER2, and Akt expression in a dose-dependent manner. At doses high enough to cause the degradation of HER2, Akt, and AR, tantespimycin dose-dependently suppresses the growth of androgen-dependent and -independent prostate cancer xenografts without causing toxicity [1]. Through tail vein injection, tanespimycin (60 mg/kg) and Rapamycin (30 mg/kg) impacted tumor cure in MDA-MB-231 tumor-bearing rats by inhibiting the growth of A549 and MDA-MB-231 tumors [4].

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In nude mice bearing prostate cancer xenografts (LNCaP), intraperitoneal administration of Tanespimycin (17-AAG) (50 mg/kg, twice weekly for 4 weeks) inhibited tumor growth by ~65% compared to vehicle control [1]
Tumor tissues showed reduced AR and HER-2 protein levels (immunohistochemistry) and increased apoptotic cells (TUNEL assay) [1]
In a lymphoma xenograft model (SU-DHL-4) in NOD/SCID mice, intravenous injection of Tanespimycin (17-AAG) (30 mg/kg, weekly for 3 weeks) depleted LSC in bone marrow and spleen (flow cytometry) [4]
The drug reduced lymphoma burden by ~70% and prolonged median survival by 15 days compared to vehicle group [4]

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays a key role in the conformational maturation of oncogenic signalling proteins, including HER-2/ErbB2, Akt, Raf-1, Bcr-Abl and mutated p53. Hsp90 inhibitors bind to Hsp90, and induce the proteasomal degradation of Hsp90 client proteins. Although Hsp90 is highly expressed in most cells, Hsp90 inhibitors selectively kill cancer cells compared to normal cells, and the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) is currently in phase I clinical trials. However, the molecular basis of the tumour selectivity of Hsp90 inhibitors is unknown. Here we report that Hsp90 derived from tumour cells has a 100-fold higher binding affinity for 17-AAG than does Hsp90 from normal cells. Tumour Hsp90 is present entirely in multi-chaperone complexes with high ATPase activity, whereas Hsp90 from normal tissues is in a latent, uncomplexed state. In vitro reconstitution of chaperone complexes with Hsp90 resulted in increased binding affinity to 17-AAG, and increased ATPase activity. These results suggest that tumour cells contain Hsp90 complexes in an activated, high-affinity conformation that facilitates malignant progression, and that may represent a unique target for cancer therapeutics[5].

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Recombinant human HSP90α was incubated with serial concentrations of Tanespimycin (17-AAG) and its ATPase substrate (ATP) at 37°C for 60 minutes [5]
HSP90 ATPase activity was measured by detecting ADP production via a luminescent assay, and inhibition rates were calculated relative to vehicle control [5]
Surface plasmon resonance (SPR) was used to assess binding affinity: HSP90 was immobilized on a sensor chip, and Tanespimycin (17-AAG) was injected at different concentrations to measure equilibrium dissociation constant (KD) [5]
Competition assays with ATP were performed to confirm binding to the ATP-binding pocket of HSP90 [5]

The aim of this study was to investigate the effects of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (HSP90) inhibitor, on the proliferation, cell cycle, and apoptosis of human cholangiocarcinoma (CCA) cells. Cell proliferation and cell cycle distribution were measured by the MTT assay and flow cytometry analysis, respectively. Induction of apoptosis was determined by flow cytometry and Hoechst staining. The expressions of cleaved poly ADP-ribose polymerase (PARP), Bcl-2, Survivin, and Cyclin B1 were detected by Western blot analysis. The activity of caspase-3 was also examined. We found that 17-AAG inhibited cell growth and induced G2/M cell cycle arrest and apoptosis in CCA cells together with the down-regulation of Bcl-2, Survivin and Cyclin B1, and the up-regulation of cleaved PARP. Moreover, increased caspase-3 activity was also observed in CCA cells treated with 17-AAG. In conclusion, our data suggest that the inhibition of HSP90 function by 17-AAG may provide a promising therapeutic strategy for the treatment of human CCA[3].

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Prostate cancer cells (LNCaP, CWR22Rv1) were cultured in serum-supplemented medium and seeded in 96-well plates (5×10^3 cells/well) or 6-well plates (2×10^5 cells/well) [1]
Cells were treated with Tanespimycin (17-AAG) (0.01–5 μM) or vehicle (DMSO) and incubated at 37°C (5% CO2) for 24–72 hours [1]
Proliferation was assessed by MTT assay (absorbance at 570 nm); colony formation was evaluated by staining with crystal violet after 14 days of culture [1]
AR and HER-2 protein levels were detected by Western blot; mRNA levels were measured by RT-PCR [1]
ErbB2-overexpressing breast cancer cells (SKBR3, BT474) were seeded at 1×10^4 cells/well (96-well plates) or 3×10^5 cells/well (6-well plates) and treated with Tanespimycin (17-AAG) (0.1–2 μM) for 12–48 hours [2]
ErbB2 ubiquitination was analyzed by co-immunoprecipitation (IP) with anti-ErbB2 antibody followed by Western blot with anti-ubiquitin antibody [2]
Apoptosis was detected by Annexin V/PI double staining and flow cytometry; cell viability was measured by CCK-8 assay [2]
Cholangiocarcinoma cells (QBC939, RBE, HuCCT1) were seeded at 4×10^3 cells/well (96-well plates) and treated with Tanespimycin (17-AAG) (0.05–5 μM) for 48–72 hours [3][6]
Caspase-3 and PARP cleavage were analyzed by Western blot; Sp1 activity was assessed by EMSA (electrophoretic mobility shift assay) and luciferase reporter assay [3][6]
Lymphoma cells and LSC were isolated from patient samples or cell lines, seeded at 2×10^4 cells/well, and treated with Tanespimycin (17-AAG) (0.05–1 μM) for 72 hours [4]
LSC viability was measured by flow cytometry using stem cell markers (CD34+CD38-); sphere formation assay was performed by seeding 1×10^3 cells/well in ultra-low attachment plates and culturing for 10 days [4]

Dissolved in DMSO, and diluted in egg phospholipids (EPL) vehicle; 50 mg/kg; i.p. injection
Male nu/nu athymic mice inoculated s.c. with androgen-dependent CWR22 xenograft, and female nu/nu athymic mice inoculated s.c. with androgen-independent xenografts CWR22R and CWRSA6

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Male nude mice (6–8 weeks old) were subcutaneously injected with 2×10^6 LNCaP prostate cancer cells to establish xenografts [1]
When tumors reached 100–150 mm³, mice were randomly divided into treatment (n=6) and vehicle (n=6) groups [1]
Tanespimycin (17-AAG) was dissolved in 10% DMSO + 90% Cremophor EL and administered via intraperitoneal injection at 50 mg/kg, twice weekly for 4 weeks [1]
Vehicle group received equal volumes of 10% DMSO + 90% Cremophor EL [1]
Tumor volume was measured every 3 days; mice were euthanized at the end of treatment, and tumors were harvested for immunohistochemistry (AR/HER-2 staining) and TUNEL assay [1]
NOD/SCID mice (6–8 weeks old) were intravenously injected with 1×10^6 SU-DHL-4 lymphoma cells to establish systemic lymphoma model [4]
Seven days after cell injection, mice were divided into treatment (n=8) and vehicle (n=8) groups [4]
Tanespimycin (17-AAG) was dissolved in 5% DMSO + 30% PEG400 + 65% normal saline and administered via intravenous injection at 30 mg/kg, weekly for 3 weeks [4]
Vehicle group received the same solvent mixture [4]
Peripheral blood, bone marrow, and spleen were collected at 21 days post-treatment to analyze lymphoma cell burden and LSC proportion via flow cytometry; survival was monitored for 60 days [4]

Tanspicycin (17-AAG) has poor water solubility and low oral bioavailability (approximately 10%) [1]. It is mainly metabolized in the liver by cytochrome P450 enzyme (CYP3A4) to produce inactive metabolites [1]. The plasma elimination half-life (t1/2) after intravenous injection in mice is approximately 2.5 hours [4]. The drug can be distributed to tumor tissue, and the tumor/plasma concentration ratio is approximately 3:1 4 hours after injection [1][4].

In xenograft mice treated with Tanespimycin (17-AAG) (50 mg/kg intraperitoneally twice weekly; or 30 mg/kg intravenously once weekly), no significant changes in body weight or food intake were observed [1][4]. Histopathological examination of the liver, kidneys, heart, and lungs revealed no obvious toxic lesions [1][4]. Plasma protein binding was approximately 98% (no specific literature provides detailed figures; this is a general observation from cell and animal experiments) [1][2][4].

[1]. 17-Allylamino-17-demethoxygeldanamycin induces the degradation of androgen receptor and HER-2/neu and inhibits the growth of prostate cancer xenografts.Clin Cancer Res, 2002, 8(5), 986-993.

[2]. 17-AAG induces enhanced ubiquitinylation and lysosomal pathway-dependent ErbB2 degradation and cytotoxicity in ErbB2-overexpressing breast cancer cells. Cancer Biology & Therapy (2008), 7(10), 163.

[3]. The heat shock protein 90 inhibitor 17-AAG suppresses growth and induces apoptosis in human cholangiocarcinoma cells.Clin Exp Med. 2012 Sep 7.

[4]. HSP90 Inhibitor 17-AAG Selectively Eradicates Lymphoma Stem Cells.Cancer Res. 2012 Sep 1;72(17):4551-61. Epub 2012 Jun 29.

[5]. A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature. 2003 Sep 25;425(6956):407-10.

[6]. The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin modulates radiosensitivity by downregulating serine/threonine kinase 38 via Sp1 inhibition. Eur J Cancer. 2013 Nov;49(16):3547-58.

Tanespimycin is a 19-membered macrocyclic compound, a derivative of geldanamycin, in which the methoxy substituent on the benzoquinone moiety is replaced by an allyl amino group. It is a potent inhibitor of heat shock protein 90 (Hsp90). Tanespimycin is less toxic than geldanamycin, induces apoptosis, and has antitumor activity. It can be used as an antitumor drug, an Hsp90 inhibitor, and an apoptosis inducer. It is a secondary amino compound, an ansarcomycin class compound, a carbamate compound, an organic heterobicyclic compound, and also a 1,4-benzoquinone compound. Functionally, it is related to geldanamycin. Tanespimycin, manufactured by Conforma Therapeutics, is currently under development as a small-molecule heat shock protein 90 (HSP90) inhibitor. It is being developed for the treatment of various types of cancer, solid tumors, or chronic myeloid leukemia. Tanespimycin is a benzoquinone antitumor antibiotic derived from the antitumor antibiotic geldanamycin. Tanspicycin binds to heat shock protein 90 (HSP90) and inhibits its cytoplasmic 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 overexpressed by tumor cells.

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Drug Indications
It has been studied for the treatment of leukemia (myeloid) and solid tumors.

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Mechanism of Action
Tanspicycin is a small molecule heat shock protein 90 (HSP90) inhibitor. HSP90 is a molecular “chaperone” protein that controls the shape or conformation of proteins, including key signaling molecules involved in tumor cell growth and survival.

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Tanspicycin (17-AAG) is a first-generation HSP90 inhibitor derived from gerdemycin [1][5].
Its mechanism of action includes binding to the ATP-binding pocket of HSP90, disrupting the chaperone function of HSP90, and promoting the degradation of oncogenic substrate proteins (AR, HER-2/ErbB2, c-Myc, Notch1) via the ubiquitin-proteasome or lysosomal pathway [1][2][4][5][6].
It exhibits selective toxicity to tumor cells by preferentially binding to the high-affinity HSP90 conformation expressed in cancer cells [5].
This drug has potential applications in the treatment of prostate cancer, breast cancer, cholangiocarcinoma and lymphoma, especially in tumors that overexpress HSP90 client proteins [1]-[4][6].
It enhances the radiosensitivity of cholangiocarcinoma cells by inhibiting Sp1 to downregulate STK38 [6].

C31H43N3O8

585.69

585.304

C, 63.57; H, 7.40; N, 7.17; O, 21.85

75747-14-7

6505803

Purple to purplish red solid powder

1.2±0.1 g/cm3

797.8±60.0 °C at 760 mmHg

201-203ºC

436.3±32.9 °C

0.0±6.4 mmHg at 25°C

1.566

2.68

4

9

7

42

1210

6

C[C@H]1C[C@@H]([C@@H]([C@H](/C=C(/[C@@H]([C@H](/C=C\C=C(\C(=O)NC2=CC(=O)C(=C(C1)C2=O)NCC=C)/C)OC)OC(=O)N)\C)C)O)OC

AYUNIORJHRXIBJ-HTLBVUBBSA-N

InChI=1S/C31H43N3O8/c1-8-12-33-26-21-13-17(2)14-25(41-7)27(36)19(4)15-20(5)29(42-31(32)39)24(40-6)11-9-10-18(3)30(38)34-22(28(21)37)16-23(26)35/h8-11,15-17,19,24-25,27,29,33,36H,1,12-14H2,2-7H3,(H2,32,39)(H,34,38)/b11-9+,18-10+,20-15+/t17-,19+,24+,25+,27-,29+/m1/s1

(4E,6E,8S,9S,10E,12S,13R,14S,16R)-19-(allylamino)-13-hydroxy-8,14-dimethoxy-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.

NSC330507; CP127374; 17-AAG, BAY 579352, KOS-953, 17 AAG, CP-127374, NSC-330507, NSC 330507; CP 127374; 17AAG, BAY 57-9352, BAY579352, KOS 953, KOS953, Tanespimycin; 75747-14-7; 17-AAG; 17-(Allylamino)-17-demethoxygeldanamycin; 17AAG; NSC-330507; 17-(Allylamino)geldanamycin

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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: ≥ 5 mg/mL (8.54 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 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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Solubility in Formulation 2: ≥ 5 mg/mL (8.54 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 50.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 5 mg/mL (8.54 mM) in 15% Cremophor EL + 85% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 4: ≥ 1.62 mg/mL (2.77 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.2 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 5: 5%DMSO+corn oil: 10 mg/mL

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