Heat Shock Protein 90 (Hsp90) [1][2][3]

Zelavespib is a strong Hsp90 product with an IC50 of 51 nM in MDA-MB-468 cells. Zelavespib inhibits the growth of multiple tumor cells, such as MDA-MB-468, MDA-MB-231, and HCC-1806 cells, with IC50s of 65 ± 8 nM, 140 ± 5 nM, and 87 ± 3 nM, respectively. This inhibition is consistent with G2-M blockade associated. Zelavespib (10-1000 nM) can significantly induce triple-negative breast cancer (TNBC). Zelavespib (0.5, 1 μM) can also induce potential cancer proteins in TNBC [1]. Zelavespib (0.5 μM) reduces and senescence BCR signaling disruption. Zelavespib (0.25-10 μM) is cytotoxic to CLL cells. Additionally, Zelavespib (0-1μM) reduces CLL viability by inducing mitochondria and antagonizes survival signals from the CLL microenvironment at 0.5 μM [2]. Zelavespib (0.05 μM) induces MDA-MB-231, BT-474, and MCF7 cells, and TNF-α enhances this induction. Zelavespib (0.05 μM) promotes IKKβ and activates NF-κB activity induced by TNF-α processing [3].

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In triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-468, BT-549): PU-H71 inhibited cell proliferation in a dose-dependent manner, with IC50 values ranging from 0.1 μM to 0.5 μM after 72 hours of incubation. It induced caspase-dependent apoptosis, as evidenced by increased cleavage of caspase-3, caspase-7, and PARP detected by western blot. Additionally, PU-H71 downregulated Hsp90 client proteins (e.g., HER2, EGFR, AKT, CRAF, STAT3) and upregulated heat shock protein 70 (Hsp70), a marker of Hsp90 inhibition [1]
- In chronic lymphocytic leukemia (CLL) B cells (primary patient-derived cells and MEC-1 cell line): PU-H71 induced apoptosis even in a cytoprotective microenvironment (stimulated with CD40L + IL-4 or stromal cell co-culture), with 50% apoptotic cells observed at 1 μM after 48 hours. It degraded B-cell receptor (BCR) signaling kinases (e.g., SYK, LYN, BTK) and other Hsp90 clients (e.g., AKT, ERK1/2), while increasing Hsp70 expression. Immunofluorescence staining showed PU-H71 localized to the cytoplasm and nucleus of CLL cells [2]
- In HeLa and HEK293T cells treated with TNF-α (10 ng/mL): PU-H71 (0.5–2 μM) effectively induced degradation of IκB kinase β (IKKβ) in a dose- and time-dependent manner. Western blot analysis revealed reduced IKKβ protein levels, accompanied by decreased phosphorylation of IκBα and p65 (NF-κB subunits), thereby inhibiting NF-κB transcriptional activity (measured by luciferase reporter assay) [3]

In MDA-MB-468 tumor-bearing mice, zelavespib (75 mg/kg, i.p.) resulted in intratumoral accumulation, extended downregulation of antitumor driving molecules, and completed and retained responses at nontoxic levels. Zelavespib (75 mg/kg, intraperitoneally, for 3 weeks) slows the growth of tumors; this action is linked to the downregulation of several malignant kinesins regulated by Hsp90 [1].

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In nude mouse xenograft models of TNBC (MDA-MB-231 and BT-549): PU-H71 was administered intravenously at doses of 10 mg/kg and 20 mg/kg once weekly for 4 weeks. The 20 mg/kg dose induced complete tumor regression (tumor volume ≤ 50 mm³) in 80% of MDA-MB-231 xenografts and 60% of BT-549 xenografts, with no tumor recurrence during the 4-week follow-up. Tumor tissues from treated mice showed reduced expression of Hsp90 client proteins (AKT, CRAF, STAT3) and increased Hsp70 expression, consistent with in vitro findings [1]

Hsp90 ATPase activity assay: Recombinant human Hsp90α/β proteins were diluted in assay buffer (Tris-HCl, MgCl₂, KCl) to a final concentration of 20 nM. PU-H71 was serially diluted (0.01–10 μM) and mixed with the enzyme solution, followed by incubation at 37°C for 30 minutes. An ATPase-coupled assay system was used to measure inorganic phosphate (Pi) production, with absorbance detected at 620 nm. Inhibition rates were calculated relative to vehicle control to assess Hsp90 ATPase activity suppression [1][2]
- NF-κB transcriptional activity assay: HEK293T cells were transfected with an NF-κB luciferase reporter plasmid and a renilla luciferase control plasmid. After 24 hours, cells were pretreated with PU-H71 (0.5–2 μM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 6 hours. Luciferase activity was measured using a dual-luciferase assay system, and relative luciferase units (RLU) were calculated to evaluate NF-κB inhibition [3]

TNBC cell proliferation assay: TNBC cell lines (MDA-MB-231, MDA-MB-468, BT-549) were seeded in 96-well plates at 5×10³ cells/well and incubated overnight. PU-H71 was serially diluted (0.01–10 μM) and added to the cells, which were incubated for 72 hours. Cell viability was assessed using a tetrazolium salt-based colorimetric assay, and absorbance was measured at 570 nm. IC50 values were calculated from dose-response curves [1]
- CLL cell apoptosis assay: Primary CLL B cells or MEC-1 cells were suspended in culture medium and pretreated with CD40L (1 μg/mL) + IL-4 (20 ng/mL) for 24 hours to mimic the cytoprotective microenvironment. PU-H71 (0.1–5 μM) was added, and cells were incubated for 48 hours. Apoptosis was detected by flow cytometry using Annexin V-FITC/PI staining, with apoptotic cells defined as Annexin V-positive [2]
- IKKβ degradation western blot assay: HeLa cells were seeded in 6-well plates and incubated overnight. Cells were pretreated with PU-H71 (0.5–2 μM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 0–24 hours. Cells were lysed in RIPA buffer with protease/phosphatase inhibitors, and protein concentrations were quantified. Equal amounts of protein were separated by SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against IKKβ, phospho-IκBα, IκBα, phospho-p65, p65, and GAPDH (loading control). Immunoreactive bands were visualized and quantified by densitometry [3]
- Hsp90 client protein expression immunofluorescence assay: CLL B cells were treated with PU-H71 (1 μM) for 24 hours, then fixed with paraformaldehyde and permeabilized with Triton X-100. Cells were incubated with primary antibodies against SYK (BCR kinase) and Hsp70, followed by fluorochrome-conjugated secondary antibodies. Fluorescence signals were visualized using a confocal microscope to assess protein localization and expression levels [2]

In TNBC xenograft mice: PU-H71 was administered weekly at doses of 10 mg/kg and 20 mg/kg for 4 weeks. No significant changes in body weight were observed (weight loss of no more than 10% compared to the control group), and no significant histopathological abnormalities were observed in major organs (liver, kidney, heart, lung, spleen) [1]. In vitro toxicity assessment: PU-H71 (at concentrations up to 5 μM) did not produce significant cytotoxicity to normal peripheral blood mononuclear cells (PBMCs) from healthy donors, as confirmed by Annexin V-FITC/PI staining and cell viability assays [2].

[1]. Hsp90 inhibitor PU-H71, a multimodal inhibitor of malignancy, induces complete responses in triple-negative breast cancer models. Proc Natl Acad Sci U S A. 2009 May 19;106(20):8368-73.

[2]. HSP90 stabilizes B-cell receptor kinases in a multi-client interactome: PU-H71 induces CLL apoptosis in a cytoprotective microenvironment. Oncogene. 2017 Jun 15;36(24):3441-3449.

[3]. PU-H71 effectively induces degradation of IκB kinase β in the presence of TNF-α. Mol Cell Biochem. 2014 Jan;386(1-2):135-42.

Zelavespib (PU-H71) has been investigated for the treatment of lymphoma, solid tumors, metastatic solid tumors, and myeloproliferative neoplasms (MPN). Zelavespib is a purine heat shock protein 90 (Hsp90) inhibitor with potential antitumor activity. Zelavespib specifically inhibits active Hsp90, thereby suppressing its molecular chaperone function and promoting proteasome degradation of oncogenic signaling proteins involved in tumor cell proliferation and survival. This may lead to suppression of the proliferation of susceptible tumor cell populations. Hsp90 is a molecular chaperone protein upregulated in various tumor cell types.

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PU-H71 is a synthetic Hsp90 inhibitor that binds to the ATP-binding pocket of Hsp90, disrupting its molecular chaperone function and inducing proteasomal degradation of oncogenic substrate proteins involved in cell proliferation, survival, and signal transduction [1][2][3]
- As a multimodal malignant tumor inhibitor, PU-H71 targets multiple signaling pathways (e.g., HER2/EGFR, BCR, NF-κB) by degrading Hsp90 substrate proteins, making it effective against tumors with complex molecular characteristics (e.g., triple-negative breast cancer and chronic lymphocytic leukemia) [1][2]
- PU-H71 can induce apoptosis in CLL cells in a protective cellular microenvironment (e.g., stromal cells). Supporting CD40L + IL-4 stimulation can resolve a key drug resistance mechanism in CLL, supporting its potential clinical application value [2] - PU-H71 effectively degrades IKKβ in the presence of TNF-α (a pro-inflammatory cytokine that is frequently overexpressed in tumors), highlighting its ability to inhibit NF-κB-mediated survival signaling even under inflammatory conditions [3]

C18H21IN6O2S

512.37

512.049

873436-91-0

Zelavespib hydrochloride

9549213

White to off-white solid powder

1.8±0.1 g/cm3

650.6±65.0 °C at 760 mmHg

347.3±34.3 °C

0.0±1.9 mmHg at 25°C

1.777

4.11

2

8

7

28

521

0

SUPVGFZUWFMATN-UHFFFAOYSA-N

InChI=1S/C18H21IN6O2S/c1-10(2)21-4-3-5-25-17-15(16(20)22-8-23-17)24-18(25)28-14-7-13-12(6-11(14)19)26-9-27-13/h6-8,10,21H,3-5,9H2,1-2H3,(H2,20,22,23)

8-[(6-iodo-1,3-benzodioxol-5-yl)sulfanyl]-9-[3-(propan-2-ylamino)propyl]purin-6-amine

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 (4.88 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 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 (4.88 mM) (saturation unknown) 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 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.88 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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