HSP90 (EC50 = 62 nM); GRP94 (EC50 = 65 nM)

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].

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].

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.

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.

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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Dissolved in DMSO; 10 mg/kg; i.p. injection

SCID mice engrafted with TCL1 leukemia cells

[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.

Alvespimycin Hydrochloride is the hydrochloride salt of alvespimycin, an analogue of the antineoplastic benzoquinone antibiotic geldanamycin. Alvespimycin binds to HSP90, a chaperone protein that aids in the assembly, maturation and folding of proteins. Subsequently, the function of Hsp90 is inhibited, leading to the degradation and depletion of its client proteins such as kinases and transcription factors involved with cell cycle regulation and signal transduction.

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.)