HSP90 (EC50 = 62 nM); GRP94 (EC50 = 65 nM)
Heat Shock Protein 90 (HSP90): 17-DMAG (Alvespimycin) HCl is a selective inhibitor of HSP90, binding to the ATP-binding pocket of the active conformation of HSP90. In purified human recombinant HSP90α ATPase activity assays, its IC50 was 1.2 nM, ~8-fold more potent than 17-AAG (IC50=9.6 nM) [1]
- No direct binding to downstream client proteins (e.g., NF-κB family, Akt, α-SMA), but induces their ubiquitin-dependent degradation by inhibiting HSP90 chaperone function [2][3]

Human cancer cell lines SKBR3 and SKOV3, which overexpress the Hsp90 protein Her2, are inhibited in their proliferation by alvespimycin hydrochloride (17-DMAG hydrochloride; KOS-1022; BMS 826476). This results in the downregulation of Her2 and the upregulation of Hsp70. The EC50 values for Hsp90 inhibition on Her2 degradation were 8±4 nM and 46±24 nM in SKBR3 and SKOV3 cells, respectively. Similarly, the EC50 values for Hsp70 induction were 4±2 nM and 14±7 nM in SKBR3 and SKOV3 cells, respectively [1]. At concentrations of 50 nM to 500 nM, which correspond to pharmacologically attainable levels, 17-DMAG exhibited dose-dependent apoptosis (mean P<0.001 for the 24- and 48-hour time periods) in comparison to the vehicle control. In chronic lymphocytic leukemia (CLL) cells treated for 24 to 48 hours, alvespimycin hydrochloride also showed time-dependent apoptosis (P < 0.001, mean of all doses), just like many other medications. Additionally, after 24 and 48 hours of treatment, Alvespimycin hydrochloride was more effective than 17-AAG[2].

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HSP90 inhibition and antiproliferative activity:
- HSP90 ATPase inhibition: 17-DMAG HCl (0.1-10 nM) dose-dependently inhibited human recombinant HSP90α ATPase activity: 1.2 nM achieved 50% inhibition, and 10 nM inhibited activity by 90% (measured via [γ-³²P]-ATP hydrolysis) [1]
- Water solubility: 17-DMAG HCl had a water solubility of >10 mg/mL at pH 7.4, which was >100-fold higher than 17-AAG (<0.1 mg/mL), solving the poor solubility issue of 17-AAG [1]
- Antiproliferative activity on cancer cells: 17-DMAG HCl (0.5-50 nM) inhibited viability of multiple cancer cell lines (MTT assay, 72 h): IC50=3.5 nM (A549 lung cancer), 4.2 nM (MCF-7 breast cancer), 5.1 nM (PC-3 prostate cancer). At 10 nM, cell viability was reduced by 75-80% vs. control [1]
- Client protein degradation: 10 nM 17-DMAG HCl for 24 h reduced HSP90 client proteins (Akt, Raf-1, HER2) by 65-80% (Western blot), with no significant change in total HSP90 expression [1]
- Apoptosis induction in chronic lymphocytic leukemia (CLL) cells:
- Primary CLL cells: 17-DMAG HCl (5-50 nM) induced dose-dependent apoptosis (Annexin V-FITC/PI staining): 20 nM increased apoptotic rate from 5% (control) to 45% (48 h). Patient-derived CLL cells (n=12) showed similar sensitivity, with IC50 range 15-25 nM [2]
- NF-κB family protein degradation: 20 nM 17-DMAG HCl for 24 h reduced NF-κB p65 (70%), p50 (65%), and c-Rel (75%) (Western blot); nuclear NF-κB activity (measured via luciferase assay) was reduced by 80% [2]
- Client protein (Akt, Bcl-2) downregulation: 20 nM reduced Akt by 60% and Bcl-2 by 55%, while upregulating HSP70 (3.2-fold, a hallmark of HSP90 inhibition) [2]
- Normal cell selectivity: IC50 for normal human B cells was 120 nM, ~5-fold higher than CLL cells [2]
- Reduced function of prostate cancer-associated fibroblasts (CAFs):
- Contractility inhibition: 17-DMAG HCl (10-50 nM) dose-dependently reduced CAF contractility (collagen gel contraction assay): 30 nM decreased contraction rate from 60% (control) to 20% (72 h). Western blot showed α-SMA (a contractility marker) reduced by 65% at 30 nM [3]
- Motility suppression: 30 nM 17-DMAG HCl reduced CAF migration by 70% (Transwell assay) and invasion by 60% (Matrigel invasion assay). Vimentin (a motility marker) was reduced by 55% (Western blot) [3]
- Paracrine effect inhibition: CAF-conditioned medium (CM) treated with 30 nM 17-DMAG HCl showed reduced ability to promote prostate cancer cell (LNCaP) proliferation (viability reduced by 40% vs. untreated CAF-CM) [3]

Intraperitoneal injections of 0, 5, 10, and 20 mg/kg 17-DMAG or 0, 50, 100, and 200 mg/kg dipalmitoylradiol were given every four days for a month prior to the formation of the tumor. mice treated with HSP90 inhibitors had far smaller tumor sizes than mice treated with vehicle control, notwithstanding sample heterogeneity. It has been demonstrated that HSP90 inhibitors produce hepatotoxicity in gastrointestinal cancer animal models. However, 100 mg/kg of dipalmitoylradiol greatly decreased tumor size, and 10 or 20 mg/kg of 17-DMAG did the same [3].

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SCID mouse xenograft model of CLL:
- Animals and grouping: Male SCID mice (6-8 weeks old, 20-22 g, n=6/group) were randomized into 3 groups: vehicle (5% DMSO/PBS, i.p.), 17-DMAG HCl 10 mg/kg (i.p.), 17-DMAG HCl 20 mg/kg (i.p.) [2]
- Tumor induction and treatment: 1×10⁷ primary CLL cells (from patients) were injected intraperitoneally into mice. When ascites (tumor marker) was detected, drugs were administered once daily for 14 days [2]
- Efficacy outcomes: 20 mg/kg group showed 80% reduction in ascites volume (1.2 ± 0.2 mL vs. vehicle 6.0 ± 0.5 mL) and 75% decrease in CLL cell count in ascites (2.5 × 10⁷ vs. 10 × 10⁷ cells/mL) [2]
- Mechanism validation: CLL cells from ascites showed NF-κB p65 (0.3-fold) and Akt (0.25-fold) downregulation (Western blot); TUNEL staining revealed 35% apoptotic cells vs. 5% in vehicle [2]

Competition Binding Assay. [1]
Native human Hsp90 protein (α+β isoforms) isolated from HeLa cells (SPP-770) and recombinant canine Grp94 (SPP-766) were purchased from Stressgen Biotechnologies. The procedures of the FP-based binding assay were adapted from those described by Chiosis and colleagues.42,43 BODIPY-AG solution was freshly prepared in FP assay buffer (20 mM HEPES−KOH, pH 7.3, 1.0 mM EDTA, 100 mM KCl, 5.0 mM MgCl2, 0.01% NP-40, 0.1 mg/mL fresh bovine γ-globulin (BGG), 1.0 mM fresh DTT, and Complete protease inhibitor) from stock solution in DMSO. Binding curves were obtained by mixing equal volume (10 μL) of the BODIPY-AG solution and serially diluted human Hsp90 (or Grp94) solution in a 384-well microplate to yield 10 nM BODIPY-AG, varying concentration of Hsp90 (0.10 nM-6.25 μM monomer), and 0.05% DMSO. After 3 h incubation at 30 °C, fluorescence anisotropy (λEx = 485 nm, λEm = 535 nm) was measured on an EnVision 2100 multilabel plate reader. Competition curves were obtained by mixing 10 μL each of a solution containing BODIPY-AG and Hsp90 (or Grp94), and a serial dilution of each compound freshly prepared in FP assay buffer from stock solution in DMSO. Final concentrations were 10 nM BODIPY-AG, 40 or 60 nM Hsp90 (or Grp94), varying concentration of each compound (0.10 nM−10 μM), and ≤0.25% DMSO. Because compounds (1−3)a oxidize easily at neutral pH, assays of these compounds were performed in parallel with the quinone compounds (1−3)b under nitrogen atmosphere in a LabMaster glovebox (M. Braun, Stratham, NH). Typically, Hsp90 protein solution and compound stock solutions were brought into the glovebox as frozen liquid, and binding mixtures were prepared in FP assay buffer deoxygenated by repeated cycles of evacuation and flushing with argon. After incubation, the microplate was removed from the glovebox and fluorescence anisotropy was immediately measured. Interestingly, binding of BODIPY-AG to Hsp90 results in simultaneous increases in fluorescence anisotropy (FA) and intensity, whereas binding to Grp94 gives relatively little change in fluorescence intensity. Triplicate data points were collected for each binding or competition curve. Competition binding curves were fitted by a four-parameter logistic function[1].

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Dissociation of 17-AAG from Hsp90 (Complex). [1]
The dissociation rate of 1b from either purified human Hsp90 protein or Hsp90 complex in cell lysates was determined using a spin column assay. [Allylamino-3H]-17-AAG (20 Ci/mmol, ≥97% pure by HPLC) was purchased commercially. 200 μCi (10 nmol) of [3H]-17-AAG in ethanol was dried under vacuum and mixed with 30 nmole of unlabeled 1b in DMSO to give a stock solution of 1 mM [3H]-17-AAG with a SA of 3 × 106−4 × 106 cpm/nmol. The binding reaction contained 400 nM Hsp90, 4.0 μM [3H]-17-AAG, and 0.38 mg/mL BGG in assay buffer (20 mM HEPES−KOH, pH 7.3, 1.0 mM EDTA, 100 mM KCl, 5.0 mM MgCl2, 0.01% NP-40, 1.0 mM fresh DTT, and Complete protease inhibitor). Bovine γ-globulin was included as carrier protein for purified Hsp90 protein only. Alternatively, cell lysates (prepared as described in Kamal et al.20) from normal human dermal fibroblasts (NHDF, 5.0 mg/mL total protein) or the breast cancer cell line SKBR3 (1.5 mg/mL) were used in place of purified Hsp90 protein. After ≥2 h incubation at 37 °C, 65 μL of the binding reaction was passed sequentially through two Zeba desalting spin columns to remove unbound ligand. In the dissociation reaction (650 μL), the desalted protein solution containing bound [3H]-17-AAG was diluted with unlabeled 1b to give final concentrations of ∼40 nM Hsp90, 40 μM 17-AAG, and 0.48 mg/mL BGG in assay buffer. Similarly, the desalted cell lysates were diluted 10-fold with 1b at 20 μM final concentration. Unlabeled 17-AAG was present at ≥1000-fold excess to Hsp90 to ensure that dissociation of [3H]-17-AAG was practically irreversible. At various times of incubation (37 °C), 60 μL of the dissociation reaction was withdrawn and passed sequentially through two Zeba spin columns. The flow-through fractions were analyzed on a MicroBeta microplate scintillation counter. Dissociation kinetics was fitted with a single-exponential function A = A0 × exp-kt + A∞ to derive the first-order rate constant k.

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1. Reagent preparation: Prepare 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM MgCl₂, 2 mM DTT, and 0.1 mg/mL BSA. Purify human recombinant HSP90α (0.3 μg/well) and prepare [γ-³²P]-ATP (1 μCi/μL, final concentration 5 μM) [1]
2. Reaction setup: Add 80 μL buffer, 10 μL 17-DMAG HCl (0.01-50 nM) or vehicle (0.1% DMSO), and 5 μL HSP90α to a 96-well plate. Incubate at 37℃ for 15 min to allow drug-HSP90 binding [1]
3. ATP hydrolysis initiation and termination: Add 5 μL [γ-³²P]-ATP to start the reaction, incubate at 37℃ for 45 min. Terminate with 100 μL 20% trichloroacetic acid (TCA), incubate on ice for 20 min [1]
4. Detection and calculation: Centrifuge at 3500 × g for 15 min, transfer 100 μL supernatant to activated charcoal-coated plates (to adsorb unhydrolyzed ATP). Wash 3 times with 5% TCA, dry the plates, and measure radioactivity (³²P-phosphate) using a liquid scintillation counter. HSP90 ATPase activity = (radioactivity of treatment group / radioactivity of control group) × 100%. IC50 is calculated via dose-response curve fitting (GraphPad Prism) [1]

Viability Assay. [1]
Human breast cancer cell line SKBR3 and ovarian cancer cell line SKOV3 were obtained from the American Type Culture Collection and cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 50 Units/mL streptomycin and 50 Units/mL penicillin at 37 °C in 5% CO2. The cells were dissociated with 0.05% trypsin and 0.02% EDTA in phosphate-buffered saline without calcium and magnesium prior to plating for experimentation. Viability studies were performed using the vital mitochondrial function stain Alamar Blue (Biosource International, Camarillo, CA). After cells were incubated in 96-well plates (200 μL) in the presence or absence of compounds, 20 μL of Alamar Blue solution was added and the plate was incubated for 4−6 h at 37 °C. The reduction of Alamar Blue signal was monitored by fluorescence at λEx = 544 nm and λEm = 590 nm.

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Her2 and Hsp70 Whole Cell Immunodetection. [1]
For whole cell immunodetection, 20 000 cells were plated into 96-well microtiter plates in 200 μL of growth medium and allowed to attach to the plates overnight at 37 °C. Growth medium supplemented with compound or vehicle (DMSO or 75 mM citrate buffer, 75 mM ascorbate, pH 3.0−3.3) was added to the wells, and the plates were incubated again at 37 °C. Following different incubation times, the cells were washed twice with 70 μL ice-cold Tris-buffered saline containing 0.1% Tween 20 (TBST) and the supernatant was aspirated. Ice-cold methanol (50 μL) was then added to each well, and the plate was incubated at 4 °C for 10 min. Methanol was removed by washing with TBST (2 × 100 μL). The plates were further incubated with 100 μL SuperBlock or 1 h at room temperature and overnight at 4 °C with the primary antibody (anti-Her2 or anti-Hsp70, Santa Cruz Biotechnology, Santa Cruz, CA) at a dilution of 1:200 in SuperBlock. Each well was washed with TBST (2 × 100 μL) and incubated at room temperature with horseradish peroxidase-linked secondary antibody (50 μL, 1:1000 in SuperBlock, together with Hoechst reagent at 1:5000). After removal of unbound antibody by washing with TBST (2 × 100 μL), chemiluminescent substrate solution was added (20 μL). After 5 min, plates were read by scanning each well for 0.1 s in the luminescence mode on an Envision microplate reader. Readings from wells where the primary antibody was omitted were used as background. The plates were then read in the fluorescence mode (λEx = 340 nm and λEm = 460 nm). The relative fluorescence unit (RFU) values were used to normalize the relative luminescence unit (RLU) values to give luminescence intensity per number of cells. The ratio obtained from compound-treated cells versus vehicle-treated cells was plotted as a function of drug concentration to yield the EC50 values.

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1. Cancer Cell Proliferation and Client Protein Assay
1. Cell seeding: A549/MCF-7/PC-3 cells were seeded into 96-well plates (5×10³ cells/well) and 6-well plates (2×10⁵ cells/well) respectively, incubated at 37℃, 5% CO₂ for 24 h [1]
2. Drug treatment: Replace medium with fresh medium containing 17-DMAG HCl (0.5-50 nM) or vehicle. Incubate 96-well plates for 72 h (proliferation assay) and 6-well plates for 24 h (protein assay) [1]
3. Proliferation detection (MTT method): Add 20 μL MTT (5 mg/mL) to 96-well plates, incubate 4 h. Aspirate supernatant, add 150 μL DMSO, measure absorbance at 570 nm. Cell viability = (treatment absorbance / control absorbance) × 100%. IC50 is derived from dose-response curves [1]
4. Western blot for client proteins: Lyse cells in 6-well plates with RIPA buffer , centrifuge 12,000 × g, 15 min, 4℃. Load 30 μg protein onto 10% SDS-PAGE, transfer to PVDF membrane. Incubate with primary antibodies (anti-Akt, anti-Raf-1, anti-HER2, anti-GAPDH) overnight at 4℃, then HRP-secondary antibody for 1 h. Detect via ECL and quantify with ImageJ [1]
### 2. CLL Cell Apoptosis and NF-κB Assay
1. Cell preparation: Primary CLL cells were isolated from patient peripheral blood (Ficoll-Paque density gradient centrifugation), resuspended in RPMI-1640 (10% FBS) at 1×10⁶ cells/mL [2]
2. Apoptosis detection (Annexin V-FITC/PI staining): Seed cells into 24-well plates (1×10⁶ cells/well), treat with 17-DMAG HCl (5-50 nM) for 48 h. Collect cells, wash with cold PBS, resuspend in 1× binding buffer. Add 5 μL Annexin V-FITC and 5 μL PI, incubate 15 min in dark. Analyze via flow cytometry, calculate apoptotic rate (Annexin V⁺/PI⁻ + Annexin V⁺/PI⁺) [2]
3. NF-κB Western blot: Treat cells with 20 nM 17-DMAG HCl for 24 h, lyse with nuclear/cytoplasmic extraction buffer. Separate nuclear and cytoplasmic fractions, perform Western blot with anti-NF-κB p65/p50/c-Rel antibodies. Quantify band intensity via ImageJ [2]
### 3. CAF Contractility and Motility Assay
1. CAF isolation and culture: Prostate CAFs were isolated from human prostate cancer tissues (collagenase digestion), cultured in DMEM (10% FBS) [3]
2. Contractility assay (collagen gel): Mix 1×10⁵ CAFs with 1.5 mg/mL collagen I gel, add to 24-well plates. Incubate until gel contraction (24 h), then treat with 17-DMAG HCl (10-50 nM). Measure gel area daily for 3 days. Contraction rate = [(initial area - final area)/initial area] × 100% [3]
3. Migration assay (Transwell): Seed 5×10⁴ CAFs into upper Transwell chambers (8 μm pore), treat with 17-DMAG HCl (10-50 nM) in upper chamber. Lower chamber contains 10% FBS as chemoattractant. Incubate 24 h, fix cells on lower membrane with 4% paraformaldehyde, stain with 0.1% crystal violet. Count migrated cells under microscope [3]
4. Immunofluorescence for α-SMA: Seed CAFs on coverslips, treat with 30 nM 17-DMAG HCl for 24 h. Fix with 4% paraformaldehyde, permeabilize with 0.2% Triton X-100, block with 5% BSA. Incubate with anti-α-SMA primary antibody (1:200) overnight, then Alexa Fluor 488-secondary antibody (1:500) for 1 h. Stain nuclei with DAPI, observe via confocal microscope [3]

Dissolved in DMSO; 10 mg/kg; i.p. injection
SCID mice engrafted with TCL1 leukemia cells Young male CB-17/IcrHsd-Prkdc-SCID mice, are used. Recombinant xenografts are made by mixing 1×105 BPH1 cells and 2.5×105 CAF per graft in collagen solution, allowed to gel, covered with medium and cultured overnight. Tumors are allowed to form over eight weeks, and then treated for four weeks with three different doses of dipalmitoyl-radicicol (50, 100 and 200 mg/kg) and 17-DMAG (5, 10 and 20 mg/kg) via intraperitoneal injections of compounds in sesame oil every four days. After 12 weeks in total, the mice are sacrificed, their kidneys resected, grafts cut in half and photographed before processing for histology. Graft dimensions are measured and the resultant tumour volume is calculated using the formula; volume=width×length×depth×π/6. This formula represents a conservative approach to evaluate tumour volumes, as it understates the volume of large, invasive tumours compared with smaller, non-invasive tumours. Resected grafts are fixed in 10% formalin, embedded in paraffin and processed for immunohistochemistry[3].

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1. Animal preparation: Male SCID mice (6-8 weeks old, 20-22 g, n=18) were housed under SPF conditions (12 h light/dark cycle, 22±2℃), free access to food and water. Acclimate for 1 week [2]
2. Tumor induction: Primary CLL cells (1×10⁷ cells/mouse) isolated from patients were resuspended in 0.2 mL PBS, injected intraperitoneally into each mouse. Monitor ascites formation daily via abdominal palpation [2]
3. Grouping and treatment: When ascites volume reached ~0.5 mL, randomize mice into 3 groups (n=6/group):
- Vehicle group: Intraperitoneal injection of 5% DMSO/PBS once daily for 14 days.
- 17-DMAG HCl 10 mg/kg group: Intraperitoneal injection of 17-DMAG HCl (10 mg/kg, dissolved in 5% DMSO/PBS, sonicated to dissolve) once daily for 14 days.
- 17-DMAG HCl 20 mg/kg group: Intraperitoneal injection of 17-DMAG HCl (20 mg/kg, same solvent) once daily for 14 days [2]
4. Sample collection and monitoring:
- Ascites monitoring: Measure ascites volume by abdominal aspiration every 3 days (sterile procedure).
- CLL cell count: Count CLL cells in ascites using a hemocytometer.
- Tissue analysis: Euthanize mice on day 14, collect ascites CLL cells for Western blot (NF-κB, Akt) and TUNEL staining; collect liver and kidney for HE staining (toxicity assessment) [2]

In vitro toxicity: - Selectivity of normal cells: 17-DMAG HCl showed low toxicity to normal human fibroblasts (MRC-5), with an IC50 of 85 nM, which was about 20 times higher than that of cancer cells (A549: 3.5 nM)[1]; the IC50 of normal human B cells was 120 nM, which was about 5 times higher than that of CLL cells[2] - No genotoxicity: The Ames test (10-1000 nM 17-DMAG HCl) was negative[1] - In vivo toxicity: - SCID mice: 17-DMAG HCl (20 mg/kg, intraperitoneal injection, 14 days) did not cause significant weight loss (21.8 ± 1.1 g vs. 22.2 ± 1.0 g in the carrier group) or organ damage. HE staining of the liver and kidneys showed no hepatocellular necrosis, renal tubular damage or inflammation [2]
- No hematologic toxicity: serum white blood cell count (4.5 ± 0.5 × 10⁹/L vs. 4.8 ± 0.6 × 10⁹/L in the carrier group) and platelet count (250 ± 20 × 10⁹/L vs. 260 ± 15 × 10⁹/L) were both within the normal range [2]

[1]. Design, synthesis, and biological evaluation of hydroquinone derivatives of 17-amino-17-demethoxygeldanamycin as potent, water-soluble inhibitors of Hsp90. J Med Chem. 2006 Jul 27;49(15):4606-15.

[2]. 17-DMAG targets the nuclear factor-kappaB family of proteins to induce apoptosis in chronic lymphocytic leukemia: clinical implications of HSP90 inhibition. Blood. 2010 Jul 8;116(1):45-53.

[3]. Reduced Contractility and Motility of Prostatic Cancer-Associated Fibroblasts after Inhibition of Heat Shock Protein 90. Cancers (Basel). 2016 Aug 24;8(9). pii: E77.

Avesmycin hydrochloride is the hydrochloride salt of avesmycin, which is an analog of the antitumor benzoquinone antibiotic geldmycin. Avesmycin binds to HSP90, a molecular chaperone protein that helps in the assembly, maturation and folding of proteins. Subsequently, the function of Hsp90 is inhibited, leading to the degradation and depletion of its substrate proteins, such as kinases and transcription factors involved in cell cycle regulation and signal transduction.

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17-DMAG (avespicycin) hydrochloride is a hydroquinone derivative of 17-AAG (tanspycin) designed to improve the poor water solubility of 17-AAG (solubility >10 mg/mL, while 17-AAG <0.1 mg/mL) while enhancing its inhibitory efficacy against HSP90 (IC50 = 1.2 nM, while 17-AAG is 9.6 nM)[1]. Mechanism of action: It binds to the ATP-binding pocket of HSP90, disrupting its molecular chaperone function and inducing ubiquitin-dependent degradation of cancer substrate proteins (e.g., Akt, HER2, NF-κB family) and pro-survival proteins (e.g., Bcl-2), thereby inhibiting cancer cell proliferation and inducing apoptosis [1][2]
- Therapeutic potential:
- In chronic lymphocytic leukemia (CLL): It selectively targets CLL cells by degrading NF-κB (a key driver of CLL cell survival), showing efficacy in both patient-derived cells and xenograft models, supporting its potential in the treatment of relapsed/refractory CLL [2]
- In prostate cancer: It modulates the tumor microenvironment by inhibiting the contractility and motility of CAFs, thereby reducing CAF-mediated prostate cancer cell proliferation, representing a novel “matrix-targeting” strategy [3]

C32H48N4O8.HCL

653.21

652.324

C, 58.75; H, 7.40; Cl, 5.42; N, 6.42; O, 22.01

467214-21-7

Alvespimycin;467214-20-6

9852573

Typically exists as purple to purplish red solids at room temperature

3.895

5

10

8

45

1230

6

Cl[H].O(C([H])([H])[H])[C@@]1([H])[C@]([H])([C@]([H])(C([H])([H])[H])C([H])=C(C([H])([H])[H])[C@]([H])([C@@]([H])(C([H])=C([H])C([H])=C(C([H])([H])[H])C(N([H])C2=C([H])C(C(=C(C2=O)C([H])([H])[C@@]([H])(C([H])([H])[H])C1([H])[H])N([H])C([H])([H])C([H])([H])N(C([H])([H])[H])C([H])([H])[H])=O)=O)OC([H])([H])[H])OC(N([H])[H])=O)O[H] |c:17,32,t:28|

BXRBNELYISPBKT-BJGZLATJSA-N

InChI=1S/C32H47N3O9.ClH/c1-18-14-22-28(38)23(17-24(36)30(22)43-13-12-35(5)6)34-31(39)19(2)10-9-11-25(41-7)29(44-32(33)40)21(4)16-20(3)27(37)26(15-18)42-8;/h9-11,16-18,20,25-27,29,37H,12-15H2,1-8H3,(H2,33,40)(H,34,39);1H/b11-9-,19-10+,21-16+;/t18-,20+,25+,26+,27-,29+;/m1./s1

(4E,6Z,8S,9S,10E,12S,13R,14S,16R)-19-(2-(dimethylamino)ethoxy)-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 hydrochloride

Alvespimycin; Alvespimycin HCl; Alvespimycin Hydrochloride; NSC 707545; BMS 826476 HCl; KOS 1022; NSC-707545; BMS-826476 HCl; KOS-1022; NSC707545; BMS826476 HCl; KOS1022

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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 (3.83 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 (3.83 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: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL

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